Transcription factor stress-related proteins and methods of use in plants

ABSTRACT

A transgenic plant transformed by a transcription factor stress-related protein (TFSRP) coding nucleic acid, wherein expression of the nucleic acid sequence in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant. Also provided are agricultural products, including seeds, produced by the transgenic plants. Also provided are isolated TFSRP, and isolated nucleic acid coding TFSRP, and vectors and host cells containing the latter. Further provided are methods of producing transgenic plants expressing TFSRP, methods of increasing expression of other genes of interest using the TFSRP, methods of identifying novel TFSRP, and methods of modifying the expression of TFSRP in plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/168,846, filed Oct. 29, 2002 and now U.S. Pat. No. 7,164,057, andthis application is copending with 11/564,883, filed Nov. 30, 2006,which is a divisional of U.S. patent application Ser. No. 10/168,846,filed Oct. 29, 2002 and now U.S. Pat. No. 7,164,057, which is anapplication filed pursuant to 35 U.S.C. § 371 that claims prioritybenefit of PCT application PCT/US00/34972, filed Dec. 22, 2000, and U.S.provisional application Ser. No. 60/171,745, filed Dec. 22, 1999, theentire contents of each of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to nucleic acid sequences encodingproteins that are associated with abiotic stress responses and abioticstress tolerance in plants. In particular, this invention relates tonucleic acid sequences encoding proteins that confer drought, cold,and/or salt tolerance to plants.

2. Background Art

Abiotic environmental stresses, such as drought stress, salinity stress,heat stress, and cold stress, are major limiting factors of plant growthand productivity. Crop losses and crop yield losses of major crops suchas rice, maize (corn) and wheat caused by these stresses represent asignificant economic and political factor and contribute to foodshortages in many underdeveloped countries.

Plants are typically exposed during their life cycle to conditions ofreduced environmental water content. Most plants have evolved strategiesto protect themselves against these conditions of desiccation. However,if the severity and duration of the drought conditions are too great,the effects on plant development, growth and yield of most crop plantsare profound. Furthermore, most of the crop plants are very susceptibleto higher salt concentrations in the soil. Continuous exposure todrought and high salt causes major alterations in the plant metabolism.These great changes in metabolism ultimately lead to cell death andconsequently yield losses.

Developing stress-tolerant plants is a strategy that has the potentialto solve or mediate at least some of these problems. However,traditional plant breeding strategies to develop new lines of plantsthat exhibit resistance (tolerance) to these types of stresses arerelatively slow and require specific resistant lines for crossing withthe desired line. Limited germplasm resources for stress tolerance andincompatibility in crosses between distantly related plant speciesrepresent significant problems encountered in conventional breeding.Additionally, the cellular processes leading to drought, cold and salttolerance in model, drought- and/or salt-tolerant plants are complex innature and involve multiple mechanisms of cellular adaptation andnumerous metabolic pathways. This multi-component nature of stresstolerance has not only made breeding for tolerance largely unsuccessful,but has also limited the ability to genetically engineer stresstolerance plants using biotechnological methods.

Therefore, what is needed is the identification of the genes andproteins involved in these multi-component processes leading to stresstolerance. Elucidating the function of genes expressed in stresstolerant plants will not only advance our understanding of plantadaptation and tolerance to environmental stresses, but also may provideimportant information for designing new strategies for crop improvement.

One model plant used in the study of stress tolerance is Arabidopsisthaliana. There are at least four different signal-transduction pathwaysleading to stress tolerance in the model plant Arabidopsis thaliana.These pathways are under the control of distinct transcription factors(Shinozaki et al., 2000 Curr. Op. Pl. Biol. 3:217-23). Regulators ofgenes, especially transcription factors, involved in these tolerancepathways are particularly suitable for engineering tolerance into plantsbecause a single gene can activate a whole cascade of genes leading tothe tolerant phenotype. Consequently, transcription factors areimportant targets in the quest to identify genes conferring stresstolerance to plants.

One transcription factor that has been identified in the prior art isthe Arabidopsis thaliana transcription factor CBF (Jaglo-Ottosen et al.,1998 Science 280:104-6). Over-expression of this gene in Arabidopsisconferred drought tolerance to this plant (Kasuga et al., 1999 NatureBiotech. 17:287-91). However, CBF is the only example to date of atranscription factor able to confer drought tolerance to plants uponover-expression.

Although some genes that are involved in stress responses in plants havebeen characterized, the characterization and cloning of plant genes thatconfer stress tolerance remains largely incomplete and fragmented. Forexample, certain studies have indicated that drought and salt stress insome plants may be due to additive gene effects, in contrast to otherresearch that indicates specific genes are transcriptionally activatedwhich leads to accumulation of new proteins in vegetative tissue ofplants under osmotic stress conditions. Although it is generally assumedthat stress-induced proteins have a role in tolerance, direct evidenceis still lacking, and the functions of many stress-responsive genes areunknown.

There is a need, therefore, to identify genes expressed in stresstolerant plants that have the capacity to confer stress resistance toits host plant and to other plant species. Newly generated stresstolerant plants will have many advantages, such as increasing the rangethat crop plants can be cultivated by, for example, decreasing the waterrequirements of a plant species.

SUMMARY OF THE INVENTION

This invention fulfills in part the need to identify new, uniquetranscription factors capable of conferring stress tolerance to plantsupon over-expression. Namely, described herein are the transcriptionfactors: 1) CAAT-Box like Binding Factor-1 (CABF-1); 2) CABF-2 3) DNABinding Factor-1 (DBF-1); 4) CRT/DRE Binding Factor (CBF-1); 5) HomeoDomain/Leucine Zipper (HDZ-1); 6) Zinc-Finger Factor (ZF-1) and 7)Leucine Zipper (LZ-1), all from Physcomitrella patens.

The present invention provides a transgenic plant transformed by atranscription factor stress-related protein (TFSRP) coding nucleic acid,wherein expression of the nucleic acid sequence in the plant results inincreased tolerance to environmental stress as compared to a wild typevariety of the plant. The invention provides that the TFSRP can beselected from one of the well known general classes of transcriptionfactor proteins: 1) CAAT-Box like Binding Factor (CABF); 2) DNA BindingFactor (DBF); 3) Homeo Domain/Leucine Zipper (HDZ); 4) Zinc-FingerFactor (ZF); and 5) Leucine Zipper (LZ). The invention further providesspecific examples of TFSRPs, and TFSRP coding nucleic acids, such as 1)CABF-1; 2) CABF-2; 3) DBF-1; 4) CRT/DRE Binding Factor (CBF-1); 5)HDZ-1; 6) ZF-1 and 7) LZ-1.

The invention provides in some embodiments that the TFSRP and codingnucleic acid are that found in members of the genus Physcomitrella. Inanother preferred embodiment, the nucleic acid and protein are from aPhyscomitrella patens plant. The invention provides that theenvironmental stress can be salinity, drought, temperature, metal,chemical, pathogenic and oxidative stresses, or combinations thereof. Inpreferred embodiments, the environmental stress can be salinity,drought, and temperature, or combinations thereof.

The invention further provides a seed produced by a transgenic planttransformed by a TFSRP coding nucleic acid, wherein the plant is truebreeding for increased tolerance to environmental stress as compared toa wild type variety of the plant. The invention further provides a seedproduced by a transgenic plant expressing a TFSRP, wherein the plant istrue breeding for increased tolerance to environmental stress ascompared to a wild type variety of the plant.

The invention further provides an agricultural product produced by anyof the above-described transgenic plants. The invention further providesan isolated TFSRP, wherein the TFSRP is as described below. Theinvention further provides an isolated TFSRP coding nucleic acid,wherein the TFSRP coding nucleic acid codes for a TFSRP as describedbelow.

The invention further provides an isolated recombinant expression vectorcomprising a TFSRP coding nucleic acid as described below, whereinexpression of the vector in a host cell results in increased toleranceto environmental stress as compared to a wild type variety of the hostcell. The invention further provides a host cell containing the vectorand a plant containing the host cell.

The invention further provides a method of producing a transgenic plantwith a TFSRP coding nucleic acid, wherein expression of the nucleic acidin the plant results in increased tolerance to environmental stress ascompared to a wild type variety of the plant comprising: (a)transforming a plant cell with an expression vector comprising a TFSRPcoding nucleic acid, and (b) generating from the plant cell a transgenicplant with an increased tolerance to environmental stress as compared toa wild type variety of the plant. In preferred embodiments, the TFSRP isas described below. In preferred embodiments, the TFSRP coding nucleicacid is as described below.

The invention further provides a method of increasing expression of agene of interest within a host cell as compared to a wild type varietyof the host cell, wherein the gene of interest is transcribed inresponse to a TFSRP, comprising: (a) transforming the host cell with anexpression vector comprising a TFSRP coding nucleic acid, and (b)expressing the TFSRP within the host cell, thereby increasing theexpression of the gene transcribed in response to the TFSRP as comparedto a wild type variety of the host cell. In preferred embodiments, theTFSRP is as described below. In preferred embodiments, the TFSRP codingnucleic acid is as described below.

The present invention further provides a method of identifying a novelTFSRP, comprising (a) raising a specific antibody response to a TFSRP,or fragment thereof, as described above; (b) screening putative TFSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel TFSRP; and(c) analyzing the bound material in comparison to known TFSRP todetermine its novelty.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A-G) show the partial cDNA sequences of CABF-1 (SEQ ID NO:1),DBF-1 (SEQ ID NO:2), CBF-1 (SEQ ID NO:3), HDZ-1 (SEQ ID NO:4), ZF-1 (SEQID NO:5), LZ-1 (SEQ ID NO:6) and CABF-2 (SEQ ID NO:7) fromPhyscomitrella patens.

FIGS. 2(A-H) show the full-length cDNA sequences of CABF-1 (SEQ IDNO:8), DBF-1 (SEQ ID NO:9), DBF-1 variant (SEQ ID NO:22), CBF-1 (SEQ IDNO:10), HDZ-1 (SEQ ID NO:11), ZF-1 (SEQ ID NO:12), LZ-1 (SEQ ID NO:13)and CABF-2 (SEQ ID NO:14) from Physcomitrella patens.

FIGS. 3(A-H) show the deduced amino acid sequences of CABF-1 (SEQ IDNO:15), DBF-1 (SEQ ID NO:16), DBF-1 variant (SEQ ID NO:23), CBF-1 (SEQID NO:17), HDZ-1 (SEQ ID NO:18), ZF-1 2 (SEQ ID NO:19), LZ-1 (SEQ IDNO:20) and CABF-2 (SEQ ID NO:21) from Physcomitrella patens.

FIG. 4 shows a diagram of the plant expression vector pGMSG containingthe super promoter driving the expression of SEQ ID NOs: 8, 9, 10, 11,12, 13, and 14 (“Desired Gene”). The components are: aacCI gentamycinresistance gene (Hajdukiewicz et al., 1994 Plant Molecular Biology25:989-94), NOS promoter (Becker et al., 1992 Plant Molecular Biology20:1195-7), g7T terminator (Becker et al., 1992), NOSpA terminator(Jefferson et al., 1987 EMBO J. 6:3901-7).

FIG. 5 shows the results of a drought stress test with over-expressingHDZ-1 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

FIG. 6 shows the results of a drought stress test with over-expressingZF-1 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

FIG. 7 shows the results of a drought stress test with over-expressingCABF-1 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

FIG. 8 shows the results of a drought stress test with over-expressingDBF-1 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

FIG. 9 shows the results of a drought stress test with over-expressingCABF-2 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

FIG. 10 shows the results of a drought stress test with over-expressingLZ-1 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

FIG. 11 shows the results of a drought stress test with over-expressingCBF-1 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

FIG. 12 shows the results of a salt stress test with over-expressingZF-1 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

FIG. 13 shows the results of a salt stress test with over-expressingCABF-2 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

FIG. 14 shows the results of a salt stress test with over-expressingLZ-1 from Physcomitrella patens in transgenic plants and wild-typeArabidopsis lines. The transgenic lines display a tolerant phenotype.Individual transformant lines are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. However, before the presentcompounds, compositions, and methods are disclosed and described, it isto be understood that this invention is not limited to specific nucleicacids, specific polypeptides, specific cell types, specific host cells,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art. It is also to be understood thatthe terminology used herein is for the purpose of describing specificembodiments only and is not intended to be limiting. In particular, thedesignation of the amino acid sequences as “Transcription FactorStress-related Proteins” (TFSRPs), in no way limits the functionality ofthose sequences.

The present invention provides a transgenic plant transformed by a TFSRPcoding nucleic acid, wherein expression of the nucleic acid sequence inthe plant results in increased tolerance to environmental stress ascompared to a wild type variety of the plant. The invention furtherprovides a seed produced by a transgenic plant transformed by a TFSRPcoding nucleic acid, wherein the seed contains the TFSRP coding nucleicacid, and wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.The invention further provides a seed produced by a transgenic plantexpressing a TFSRP, wherein the seed contains the TFSRP, and wherein theplant is true breeding for increased tolerance to environmental stressas compared to a wild type variety of the plant. The invention furtherprovides an agricultural product produced by any of the above-orbelow-described transgenic plants. As used herein, the term “variety”refers to a group of plants within a species that share constantcharacters that separate them from the typical form and from otherpossible varieties within that species. While possessing at least onedistinctive trait, a variety is also characterized by some variationbetween individuals within the variety, based primarily on the Mendeliansegregation of traits among the progeny of succeeding generations. Avariety is considered “true breeding” for a particular trait if it isgenetically homozygous for that trait to the extent that, when thetrue-breeding variety is self-pollinated, a significant amount ofindependent segregation of the trait among the progeny is not observed.In the present invention, the trait arises from the transgenicexpression of a single DNA sequence introduced into a plant variety.

The invention further provides an isolated TFSRP. The invention providesthat the TFSRP can be selected from one of the well known generalclasses of transcription factor proteins, such as: 1) CAAT-Box likeBinding Factor (CABF); 2) DNA Binding Factor (DBF); 3) HomeoDomain/Leucine Zipper (HDZ); 4) Zinc-Finger Factor (ZF); and 5) LeucineZipper (LZ). It is a novel finding of the present invention that theseclasses of transcription factors are involved in stress tolerance inplants and that expression of a member of one of these protein classesin a plant can increase that plant's tolerance to stress. In furtherpreferred embodiments, the TFSRP is selected from 1) a CAAT-Box likeBinding Factor-1 (CABF-1) as defined in SEQ ID NO:15; 2) a CABF-2 asdefined in SEQ ID NO:21; 3) a DNA Binding Factor-1 (DBF-1) as defined inSEQ ID NO:16; 4) a CRT/DRE Binding Factor (CBF-1) as defined in SEQ IDNO:17; 5) a Homeo Domain/Leucine Zipper (HDZ-1) as defined in SEQ IDNO:18; 6) a Zinc-Finger Factor (ZF-1) as defined in SEQ ID NO:19; 7) aLeucine Zipper (LZ-1) as defined in SEQ ID NO:20; 8) a DNA BindingFactor-1 variant (DBF-1 v) as defined in SEQ ID NO:23 and homologuesthereof. Homologues of the amino acid sequences are defined below.

The invention further provides an isolated TFSRP coding nucleic acid.The present invention includes TFSRP coding nucleic acids that encodeTFSRPs as described herein. In preferred embodiments, the TFSRP codingnucleic acid is selected from 1) a CAAT-Box like Binding Factor-1(CABF-1) as defined in SEQ ID NO:1; 2) a CABF-2 as defined in SEQ IDNO:7; 3) a DNA Binding Factor-1 (DBF-1) as defined in SEQ ID NO:2; 4) aCRT/DRE Binding Factor (CBF-1) as defined in SEQ ID NO:3; 5) a HomeoDomain/Leucine Zipper (HDZ-1) as defined in SEQ ID NO:4; 6) aZinc-Finger Factor (ZF-1) as defined in SEQ ID NO:5; 7) a Leucine Zipper(LZ-1) as defined in SEQ ID NO:6; a DNA Binding Factor-1 variant (DBF-1v) as defined in SEQ ID NO:22 and homologues thereof. Homologues of thenucleotide sequences are defined below. In one preferred embodiment, thenucleic acid and protein are isolated from the plant genusPhyscomitrella. In another preferred embodiment, the nucleic acid andprotein are from a Physcomitrella patens (P. patens) plant.

As used herein, the term “environmental stress” refers to anysub-optimal growing condition and includes, but is not limited to,sub-optimal conditions associated with salinity, drought, temperature,metal, chemical, pathogenic and oxidative stresses, or combinationsthereof. In preferred embodiments, the environmental stress can besalinity, drought, and temperature, or combinations thereof, and inparticular, can be high salinity, low water content and low temperature.It is also to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized.

In accordance with the purposes of this invention, as embodied andbroadly described herein, this invention, in one aspect, provides anisolated nucleic acid from a moss encoding a Stress-related Protein(SRP), or a portion thereof. In particular, the present inventionprovides nucleic acids encoding TFSRPs including the nucleic acidsequences shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:22. The presentinvention also provides amino acid sequences of TFSRPs including theamino acid sequences shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23.

The present invention describes for the first time the predicted P.patens proteins CABF-1 (SEQ ID NO:15) and CABF-2 (SEQ ID NO:21) that arehomologous to CAAT-Box Binding Factors. (Homology to other proteins isshown in Tables 4 and 6, respectively). The amino acid sequence ofCABF-1 (SEQ ID NO:15) is similar to the domain “B” of other CAAT-BoxBinding Factors (Johnson and McKnight, 1989 Ann. Rev. Biochem.58:799-840). In general, CABFs are members of multi-componenttranscription activation complex. They are involved as generaltranscriptional regulators as well as in the activation of specificgenes. The particular combination of the different CABFs and othersub-units determines which genes are targeted and activated. The presentinvention also describes for the first time that CABF proteins such asCABF-1 (SEQ ID NO:15), are useful for increasing stress tolerance inplants. Particularly, the present invention demonstrates that CABF-1 isimportant for the activation of drought-related genes upon expression inArabidopsis thaliana.

Another novel predicted P. patens protein described herein is DBF-1 (SEQID NO:16), which is homologous to several eukaryotic proteins implicatedin gene regulation (transcription factors) and/or chromatin structuremodulation (i.e. helicases), for example the gene Etl-1 from mouse(Soininen et al. 1992 Mech Dev. 39:111-23). (Homology to other proteinsis shown in Table 5). The identity between DBF-1 (SEQ ID NO:16) andEtl-1 is greater in the C-terminus of the later; a region where theidentity with other known transcription factors and/or helicases(chromatin-structure changing proteins) is the greatest. Hence, DBF-1(SEQ ID NO:16) contains the functional domains of these other proteins,a fact that strengthens the hypothesis that this protein functions invivo. Over-expression of DBF-1 in Arabidopsis thaliana permits for theconstitutive, strong expression of drought-related genes in this plant,and results in a drought tolerant plant. Interestingly, there seem to betwo specifically observed variant forms of protein DBF-1, SEQ ID NO:16and SEQ ID NO:23, in P. patens and both variants are equally efficientin conferring stress tolerance to a transgenic plant.

Another novel predicted P. patens protein described herein is CBF-1 (SEQID NO:17), which is a homologue of the Arabidopsis thalianatranscription factor CBF-1. (Homology to other proteins is shown inTable 8). As mentioned before, expression of CBF-1 leads tostress-tolerant plants. Because CBF-1 (SEQ ID NO:17) originates from astress-tolerant plant, Physcomitrella patens, it is conceivable thatthis gene confers a higher level of stress tolerance to other plantsthan the Arabidopsis homologue.

Yet another discovery of the present invention is that a group ofHomeodomain/Leucine Zipper transcription factors confer increased stresstolerance to plants. Also described is a novel predicted P. patentsprotein designated HDZ-1 (SEQ ID NO:18), which is a homologue of HD-Ztranscription factors found in plants. (Homology to other proteins isshown in Table 2). Homeodomain (HD) transcription factors have been wellcharacterized in animals as being involved in organ formation. Inplants, HD proteins seem to contain, in many cases, an adjacent LeucineZipper domain (HD-Z proteins). Most of these genes are specificallyexpressed in meristems; consistent with their role in morphologydetermination (Tornero et al., 1996 Pl. J. 9:639-48). However, HD-Zproteins have also been implicated in non-developmental processes.Expression of HDZ-1 (SEQ ID NO:18) in Arabidopsis thalianaconstitutively activates genes involved in drought tolerance, resultingin drought-tolerant plants.

Another novel predicted P. patens protein described herein is ZF-1 (SEQID NO:19), which shows sequence similarity to the Zinc-Finger class oftranscription factors. (Homology to other proteins is shown in Table 3).Zinc-finger transcription factors share a specific secondary structurewhere a zinc molecule is sequestered via its interaction with cysteineor histidine amino acid residues. Through these “fingers,” the proteinsinteract with their specific DNA targets. After binding, they regulatetranscription of the target genes. Zinc-finger factors are associated inyeast with the regulation of multiple genes, e.g., genes involved ingeneral metabolism. In plants, a zinc-finger protein, CONSTANS, isresponsible for determining flowering time (Putterill et al., 1995 Cell80:847-57). The present invention also describes for the first time thatZF transcription factors are useful for increasing stress tolerance inplants. Particularly, the present invention demonstrates that ZF-1 fromP. patens is important for the activation of drought-related genes uponexpression in Arabidopsis thaliana.

Another novel predicted protein described herein is LZ-1 (SEQ ID NO:20),which shares amino acid sequence similarity with other Leucine-Zippertranscription factors (Ehrlich et al., 1992 Gene 15: 169-78). (Homologyto other proteins is shown in Table 7). Leucine-Zipper transcriptionfactors are also involved in numerous other processes in the life cycleof a plant; ranging from light-specific gene expression to seed-specificgene induction. The present invention described for the first time thatLZ transcription factors confer stress tolerance to transgenic plants,and in particular that LZ-1 from P. patens confers stress tolerance toArabidopsis thaliana plants.

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode TFSRP polypeptides or biologically active portions thereof,as well as nucleic acid fragments sufficient for use as hybridizationprobes or primers for the identification or amplification ofTFSRP-encoding nucleic acid (e.g., TFSRP DNA). As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 1000 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 200 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one that is substantially separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid. Preferably,an “isolated” nucleic acid is free of some of the sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated TFSRP nucleic acid molecule can contain less than about 5kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequenceswhich naturally flankthe nucleic acid molecule in genomic DNA of thecell from which the nucleic acid is derived (e.g., a Physcomitrellapatens cell). Moreover, an “isolated” nucleic acid molecule, such as acDNA molecule, can be free from some of the other cellular material withwhich it is naturally associated, or culture medium when produced byrecombinant techniques, or chemical precursors or other chemicals whenchemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence of SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQID NO:22, or a portion thereof, can be isolated using standard molecularbiology techniques and the sequence information provided herein. Forexample, a P. patens TFSRP cDNA can be isolated from a P. patens libraryusing all or portion of one of the sequences of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ IDNO:7 as a hybridization probe and standard hybridization techniques(e.g., as described in Sambrook et al., 1989 Molecular Cloning. ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.). Moreover, a nucleicacid molecule encompassing all or a portion of one of the sequences ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, and SEQ ID NO:7 can be isolated by the polymerase chain reactionusing oligonucleotide primers designed based upon this sequence (e.g., anucleic acid molecule encompassing all or a portion of one of thesequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, and SEQ ID NO:7 can be isolated by the polymerasechain reaction using oligonucleotide primers designed based upon thissame sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, or SEQ ID NO:7). For example, mRNA can be isolatedfrom plant cells (e.g., by the guanidinium-thiocyanate extractionprocedure of Chirgwin et al., 1979 Biochemistry 18:5294-5299) and cDNAcan be prepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for polymerase chain reactionamplification can be designed based upon one of the nucleotide sequencesshown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 or SEQ ID NO:7. A nucleic acid molecule of theinvention can be amplified using cDNA or, alternatively, genomic DNA, asa template and appropriate oligonucleotide primers according to standardPCR amplification techniques. The nucleic acid molecule so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to a TFSRPnucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences shown in SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14 or SEQ ID NO:22. The sequences of SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14 or SEQ ID NO:22 correspond to the Physcomitrella patens TFSRPcDNAs of the invention. These cDNAs comprise sequences encoding TFSRPs(i.e., the “coding region”, indicated in Table 1), as well as 5′untranslated sequences and 3′ untranslated sequences. It is to beunderstood that SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:22 comprise bothcoding regions and 5′ and 3′ untranslated regions. Alternatively, thenucleic acid molecule can comprise only the coding region of any of thesequences in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:22 or can containwhole genomic fragments isolated from genomic DNA. A coding region ofthese sequences is indicated as “ORF position”. It is to be understoodthat the

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofone of the nucleotide sequences shown in SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQID NO:22, or a portion thereof. A nucleic acid molecule which iscomplementary to one of the nucleotide sequences shown in SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14 or SEQ ID NO:22 is one which is sufficiently complementary toone of the nucleotide sequences shown in SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQID NO:22 such that it can hybridize to one of the nucleotide sequencesshown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:22, thereby forming astable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a nucleotide sequence which is at least about50-60%, preferably at least about 60-70%, more preferably at least about70-80%, 80-90%, or 90-95%, and even more preferably at least about 95%,96%, 97%, 98%, 99% or more homologous to a nucleotide sequence shown inSEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14 or SEQ ID NO:22, or a portion thereof. In anadditional preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, to one of the nucleotidesequences shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:22, or a portionthereof. These hybridization conditions include washing with a solutionhaving a salt concentration of about 0.02 molar at pH 7 at about 60° C.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences in SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14 and SEQ ID NO:22, for example a fragment which can be used as aprobe or primer or a fragment encoding a biologically active portion ofa TFSRP. The nucleotide sequences determined from the cloning of theTFSRP genes from P. patens allows for the generation of probes andprimers designed for use in identifying and/or cloning TFSRP homologuesin other cell types and organisms, as well as TFSRP homologues fromother mosses or related species. Therefore this invention also providescompounds comprising the nucleic acid molecules disclosed herein, orfragments thereof. These compounds include the nucleic acid moleculesattached to a moiety. These moieties include, but are not limited to,detection moieties, hybridization moieties, purification moieties,delivery moieties, reaction moieties, binding moieties, and the like.The probe/primer typically comprises substantially isolatedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50 or 75consecutive nucleotides of a sense strand of one of the sequences setforth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:22, an anti-sensesequence of one of the sequences set forth in SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 andSEQ ID NO:22, or naturally occurring mutants thereof. Primers based on anucleotide sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:22 can beused in PCR reactions to clone TFSRP homologues. Probes based on theTFSRP nucleotide sequences can be used to detect transcripts or genomicsequences encoding the same or homologous proteins. In preferredembodiments, the probe further comprises a label group attached thereto,e.g. the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as a part of agenomic marker test kit for identifying cells which express an TFSRP,such as by measuring a level of a TFSRP-encoding nucleic acid in asample of cells, e.g., detecting TFSRP mRNA levels or determiningwhether a genomic TFSRP gene has been mutated or deleted.

In particular, a useful method to ascertain the level of transcriptionof the gene (an indicator of the amount of mRNA available fortranslation to the gene product) is to perform a Northern blot (forreference see, for example, Ausubel et al., 1988 Current Protocols inMolecular Biology, Wiley: New York), in which a primer designed to bindto the gene of interest is labeled with a detectable tag (usuallyradioactive or chemiluminescent), such that when the total RNA of aculture of the organism is extracted, run on gel, transferred to astable matrix and incubated with this probe, the binding and quantity ofbinding of the probe indicates the presence and also the quantity ofmRNA for this gene. This information at least partially demonstrates thedegree of transcription of the transformed gene. Total cellular RNA canbe prepared from cells, tissues or organs by several methods, allwell-known in the art, such as that described in Bormann, E. R. et al.,1992 Mol. Microbiol. 6:317-326.

To assess the presence or relative quantity of protein translated fromthis mRNA, standard techniques, such as a Western blot, may be employed(see, for example, Ausubel et al., 1988 Current Protocols in MolecularBiology, Wiley: New York). In this process, total cellular proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose, and incubated with a probe, such as an antibody,which specifically binds to the desired protein. This probe is generallytagged with a chemiluminescent or colorimetric label that may be readilydetected. The presence and quantity of label observed indicates thepresence and quantity of the desired mutant protein present in the cell.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid sequence of SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21 or SEQ ID NO:23 such that the protein or portion thereofmaintains the same or a similar function as the amino acid sequence towhich it is compared. As used herein, the language “sufficientlyhomologous” refers to proteins or portions thereof which have amino acidsequences which include a minimum number of identical or equivalent(e.g., an amino acid residue which has a similar side chain as an aminoacid residue in one of the ORFs of a sequence of SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21 or SEQ ID NO:23) amino acid residues to a TFSRP amino acidsequence such that the protein or portion thereof is able to participatein a stress tolerance response in a plant, or more particularly canparticipate in the transcription of a protein involved in a stresstolerance response in a Physcomitrella patens plant. Examples of suchactivities are also described herein. Examples of TFSRP activities areset forth in Table 1.

In another embodiment, the protein is at least about 50-60%, preferablyat least about 60-70%, and more preferably at least about 70-80%,80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% ormore homologous to an entire amino acid sequence shown in SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21 or SEQ ID NO:23. In yet another embodiment, at least about50-60%, preferably at least about 60-70%, and more preferably at leastabout 70-80%, 80-90%, 90-95%, and most preferably at least about 96%,97%, 98%, 99% or more homologous to an entire amino acid sequenceencoded by a nucleic acid sequence shown in SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 orSEQ ID NO:22.

Portions of proteins encoded by the TFSRP nucleic acid molecules of theinvention are preferably biologically active portions of one of theTFSRPs. As used herein, the term “biologically active portion of aTFSRP” is intended to include a portion, e.g., a domain/motif, of aTFSRP that participates in a stress tolerance response in a plant, ormore particularly participates in the transcription of a proteininvolved in a stress tolerance response in a plant, or has an activityas set forth in Table 1. To determine whether a TFSRP or a biologicallyactive portion thereof can participate in transcription of a proteininvolved in a stress tolerance response in a plant, a stress analysis ofa plant expressing the TFSRP may be performed. Such analysis methods arewell known to those skilled in the art, as detailed in Example 7.

Additional nucleic acid fragments encoding biologically active portionsof a TFSRP can be prepared by isolating a portion of one of thesequences in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23, expressing theencoded portion of the TFSRP or peptide (e.g., by recombinant expressionin vitro) and assessing the activity of the encoded portion of the TFSRPor peptide.

The invention further encompasses nucleic acid molecules that differfrom one of the nucleotide sequences shown in SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 orSEQ ID NO:22 (and portions thereof) due to degeneracy of the geneticcode and thus encode the same TFSRP as that encoded by the nucleotidesequences shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:22. In a furtherembodiment, the nucleic acid molecule of the invention encodes a fulllength Physcomitrella patens protein which is substantially homologousto an amino acid sequence of a polypeptide shown in SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21 or SEQ ID NO:23.

In addition to the Physcomitrella patens TFSRP nucleotide sequencesshown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:22, it will beappreciated by those skilled in the art that DNA sequence polymorphismsthat lead to changes in the amino acid sequences of TFSRPs may existwithin a population (e.g., the Physcomitrella patens population). Suchgenetic polymorphism in the TFSRP gene may exist among individualswithin a population due to natural variation. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules comprisingan open reading frame encoding a TFSRP, preferably a Physcomitrellapatens TFSRP. Such natural variations can typically result in 1-5%variance in the nucleotide sequence of the TFSRP gene. Any and all suchnucleotide variations and resulting amino acid polymorphisms in a TFSRPthat are the result of natural variation and that do not alter thefunctional activity of the TFSRPs are intended to be within the scope ofthe invention.

Nucleic acid molecules corresponding to natural variants andnon-Physcomitrella patens homologues of the Physcomitrella patens TFSRPcDNA of the invention can be isolated based on their homology toPhyscomitrella patens TFSRP nucleic acid disclosed herein using thePhyscomitrella patens cDNA, or a portion thereof, as a hybridizationprobe according to standard hybridization techniques under stringenthybridization conditions. Accordingly, in another embodiment, anisolated nucleic acid molecule of the invention is at least 15nucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14 or SEQ ID NO:22. In other embodiments, the nucleic acid is atleast 30, 50, 100, 250 or more nucleotides in length. As used herein,the term “hybridizes under stringent conditions” is intended to describeconditions for hybridization and washing under which nucleotidesequences at least 60% homologous to each other typically remainhybridized to each other. Preferably, the conditions are such thatsequences at least about 65%, more preferably at least about 70%, andeven more preferably at least about 75% or more homologous to each othertypically remain hybridized to each other. Such stringent conditions areknown to those skilled in the art and can be found in Current Protocolsin Molecular Biology, 6.3.1-6.3.6, John Wiley & Sons, N.Y. (1989). Apreferred, non-limiting example of stringent hybridization conditionsare hybridization in 6× sodium chloride/sodium citrate (SSC) at about45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to a sequence of SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14 or SEQ ID NO:22 corresponds to a naturally occurring nucleic acidmolecule. As used herein, a “naturally-occurring” nucleic acid moleculerefers to an RNA or DNA molecule having a nucleotide sequence thatoccurs in nature (e.g., encodes a natural protein). In one embodiment,the nucleic acid encodes a natural Physcomitrella patens TFSRP.

In addition to naturally-occurring variants of the TFSRP sequence thatmay exist in the population, the skilled artisan will further appreciatethat changes can be introduced by mutation into a nucleotide sequence ofSEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14 or SEQ ID NO:22, thereby leading to changes inthe amino acid sequence of the encoded TFSRP, without altering thefunctional ability of the TFSRP. For example, nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues can be made in a sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ IDNO:22. A “non-essential”amino acid residue is a residue that can bealtered from the wild-type sequence of one of the TFSRPs (SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21 and SEQ ID NO:23) without altering the activity of saidTFSRP, whereas an “essential” amino acid residue is required for TFSRPactivity. Other amino acid residues, however, (e.g., those that are notconserved or only semi-conserved in the domain having TFSRP activity)may not be essential for activity and thus are likely to be amenable toalteration without altering TFSRP activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding TFSRPs that contain changes in amino acid residuesthat are not essential for TFSRP activity. Such TFSRPs differ in aminoacid sequence from a sequence contained in SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 orSEQ ID NO:23, yet retain at least one of the TFSRP activities describedherein. In one embodiment, the isolated nucleic acid molecule comprisesa nucleotide sequence encoding a protein, wherein the protein comprisesan amino acid sequence at least about 50% homologous to an amino acidsequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23 and is capable ofparticipating in the a stress tolerance response in a plant, or moreparticularly participates in the transcription of a protein involved ina stress tolerance response in a Physcomitrella patens plant, or has oneor more activities set forth in Table 1. Preferably, the protein encodedby the nucleic acid molecule is at least about 50-60% homologous to oneof the sequences of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23, morepreferably at least about 60-70% homologous to one of the sequences ofSEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23, even more preferably atleast about 70-80%, 80-90%, 90-95% homologous to one of the sequences ofSEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23, and most preferably at leastabout 96%, 97%, 98%, or 99% homologous to one of the sequences of SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21 or SEQ ID NO:23.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23 and amutant form thereof) or of two nucleic acids, the sequences are alignedfor optimal comparison purposes (e.g., gaps can be introduced in thesequence of one protein or nucleic acid for optimal alignment with theother protein or nucleic acid). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in one sequence (e.g., one of the sequences ofSEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23) is occupied by the sameamino acid residue or nucleotide as the corresponding position in theother sequence (e.g., a mutant form of the sequence selected from thepolypeptide of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23), then themolecules are homologous at that position (i.e., as used herein aminoacid or nucleic acid “homology” is equivalent to amino acid or nucleicacid “identity”). The percent homology between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % homology=numbers of identical positions/total numbers ofpositions×100). Preferably, the length of sequence comparison is atleast 15 amino acid residues, more preferably at least 25 amino acidresidues, and most preferably at least 35 amino acid residues.

Alternatively, a determination of the percent homology between twosequences can be accomplished using a mathematical algorithm. Apreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of two sequences is the algorithm of Karlin and Altschul(1990 Proc. Natl. Acad. Sci. USA 90:5873-5877). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990 J. Mol. Biol. 215:403-410). BLAST nucleic acid searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleic acid sequences homologous to TFSRP nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to TFSRPs of the present invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997 Nucleic Acids Res. 25:3389-3402).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.Another preferred non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller (CABIOS 1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) that is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of 4 can be used to obtain amino acid sequenceshomologous to the TFSRPs of the present invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997 Nucleic Acids Res. 25:3389-3402).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used.Another preferred non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller (CABIOS 1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0) that is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of 4 can be used.

An isolated nucleic acid molecule encoding a TFSRP homologous to aprotein sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23 can becreated by introducing one or more nucleotide substitutions, additionsor deletions into a nucleotide sequence of SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQID NO:22 such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced into one of the sequences of SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ IDNO:22 by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a TFSRPis preferably replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of a TFSRP coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for a TFSRP activity described herein to identify mutants thatretain TFSRP activity. Following mutagenesis of one of the sequences ofSEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14 or SEQ ID NO:22, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined by analyzing the stress tolerance of a plant expressing theprotein as described in Example 7.

In addition to the nucleic acid molecules encoding TFSRPs describedabove, another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense thereto. An “antisense” nucleic acidcomprises a nucleotide sequence that is complementary to a “sense”nucleic acid encoding a protein, e.g., complementary to the codingstrand of a double-stranded cDNA molecule or complementary to an mRNAsequence. Accordingly, an antisense nucleic acid can hydrogen bond to asense nucleic acid. The antisense nucleic acid can be complementary toan entire TFSRP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a TFSRP.The term “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the entire coding region of . . . comprises nucleotides 1 to . . . ). Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding TFSRP. The term “noncoding region” refers to 5′ and 3′sequences that flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding TFSRP disclosed herein (e.g.,the sequences set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:22),antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of TFSRP mRNA,but more preferably is an oligonucleotide which is antisense to only aportion of the coding or noncoding region of TFSRP mRNA. For example,the antisense oligonucleotide can be complementary to the regionsurrounding the translation start site of TFSRP mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in length. An antisense nucleic acid of theinvention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a TFSRP tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic (includingplant) promoters are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al., 1987 Nucleic Acids. Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., 1987 Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987 FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach, 1988 Nature 334:585-591)) can be used to catalytically cleaveTFSRP mRNA transcripts to thereby inhibit translation of TFSRP mRNA. Aribozyme having specificity for a TFSRP-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a TFSRP cDNA disclosedherein (i.e., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:22) or on the basis ofa heterologous sequence to be isolated according to methods taught inthis invention. For example, a derivative of a Tetrahymena L-19 IVS RNAcan be constructed in which the nucleotide sequence of the active siteis complementary to the nucleotide sequence to be cleaved in anTFSRP-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 andCech et al. U.S. Pat. No. 5,116,742. Alternatively, TFSRP mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W.,1993 Science 261:1411-1418.

Alternatively, TFSRP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a TFSRPnucleotide sequence (e.g., a TFSRP promoter and/or enhancer) to formtriple helical structures that prevent transcription of an TFSRP gene intarget cells. See generally, Helene, C., 1991 Anticancer Drug Des.6(6):569-84; Helene, C. et al., 1992 Ann. N.Y. Acad. Sci. 660:27-36; andMaher, L. J., 1992 Bioassays 14(12):807-15.

The invention further provides an isolated recombinant expression vectorcomprising a nucleic acid as described above, wherein expression of thevector in a host cell results in increased tolerance to environmentalstress as compared to a wild type variety of the host cell. As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) or see:Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnology, eds. Glick and Thompson, Chapter 7, 89-108, CRC Press:Boca Raton, Fla., including the references therein. Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells or under certainconditions. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., TFSRPs, mutant forms of TFSRPs, fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of TFSRPs in prokaryotic or eukaryotic cells. For example,TFSRP genes can be expressed in bacterial cells such as C. glutamicum,insect cells (using baculovirus expression vectors), yeast and otherfungal cells (see Romanos, M. A. et al., 1992 Foreign gene expression inyeast: a review, Yeast 8:423-488; van den Hondel, C. A. M. J. J. et al.,1991 Heterologous gene expression in filamentous fungi, in: More GeneManipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p. 396-428:Academic Press: San Diego; and van den Hondel, C. A. M. J. J. & Punt, P.J., 1991 Gene transfer systems and vector development for filamentousfungi, in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al.,eds., p. 1-28, Cambridge University Press: Cambridge), algae (Falciatoreet al., 1999 Marine Biotechnology 1(3):239-251), ciliates of the types:Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena,Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in WO 98/01572 and multicellular plant cells (seeSchmidt, R. and Willmitzer, L., 1988 High efficiency Agrobacteriumtumefaciens-mediated transformation of Arahidopsis thaliana leaf andcotyledon explants, Plant Cell Rep. 583-586); Plant Molecular Biologyand Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, S.71-119(1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung und R.Wu, 128-43, Academic Press: 1993; Potrykus, 1991 Annu. Rev. PlantPhysiol. Plant Molec. Biol. 42:205-225 and references cited therein) ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology. Methods in Enzymology 185, Academic Press:San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S., 1988 Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. In oneembodiment, the coding sequence of the TFSRP is cloned into a pGEXexpression vector to create a vector encoding a fusion proteincomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X protein. The fusion protein can be purified by affinitychromatography using glutathione-agarose resin. Recombinant TFSRPunfused to GST can be recovered by cleavage of the fusion protein withthrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988 Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology. Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by aco-expressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in the bacterium chosen for expression, such asC. glutamicum (Wada et al., 1992 Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

In another embodiment, the TFSRP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., 1987 Embo J. 6:229-234), pMFa (Kurjanand Herskowitz, 1982 Cell 30:933-943), pJRY88 (Schultz et al., 1987 Gene54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.).Vectors and methods for the construction of vectors appropriate for usein other fungi, such as the filamentous fungi, include those detailedin: van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) “Gene transfersystems and vector development for filamentous fungi, in: AppliedMolecular Genetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28,Cambridge University Press: Cambridge.

Alternatively, the TFSRPs of the invention can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., 1983 Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow and Summers, 1989 Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B., 1987 Nature329:840) and pMT2PC (Kaufman et al., 1987 EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987 Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988 Adv. Immunol. 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore, 1989 EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983 Cell 33:729-740; Queen andBaltimore, 1983 Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989 PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al., 1985 Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss, 1990 Science 249:374-379)and the fetoprotein promoter (Campes and Tilghman, 1989 Genes Dev.3:537-546).

In another embodiment, the TFSRPs of the invention may be expressed inunicellular plant cells (such as algae) (see Falciatore et al., 1999Marine Biotechnology 1(3):239-251 and references therein) and plantcells from higher plants (e.g., the spermatophytes, such as cropplants). Examples of plant expression vectors include those detailed in:Becker, D., Kemper, E., Schell, J. and Masterson, R., 1992 New plantbinary vectors with selectable markers located proximal to the leftborder, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W., 1984 BinaryAgrobacterium vectors for plant transformation, Nucl. Acid. Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; in: TransgenicPlants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu,Academic Press, 1993, S. 15-38.

A plant expression cassette preferably contains regulatory sequencescapable of driving gene expression in plants cells and which areoperably linked so that each sequence can fulfill its function, forexample, termination of transcription by polyadenylation signals.Preferred polyadenylation signals are those originating fromAgrobacterium tumefaciens t-DNA such as the gene 3 known as octopinesynthase of the Ti-plasmid pTiACH5 (Gielen et al., 1984 EMBO J. 3:835)or functional equivalents thereof but also all other terminatorsfunctionally active in plants are suitable.

As plant gene expression is very often not limited on transcriptionallevels, a plant expression cassette preferably contains other operablylinked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.,1987 Nucl. Acids Research 15:8693-8711).

Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CAMV (Franck et al., 1980 Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5,352,605 and WO8402913) or plant promoters likethose from Rubisco small subunit described in U.S. Pat. No. 4,962,028.

Other preferred sequences for use in plant gene expression cassettes aretargeting-sequences necessary to direct the gene product in itsappropriate cell compartment (for review see Kermode, 1996 Crit. Rev.Plant Sci. 15(4):285-423 and references cited therein) such as thevacuole, the nucleus, all types of plastids like amyloplasts,chloroplasts, chromoplasts, the extracellular space, mitochondria, theendoplasmic reticulum, oil bodies, peroxisomes and other compartments ofplant cells.

Plant gene expression can also be facilitated via an inducible promoter(for review see Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol. Biol.48:89-108). Chemically inducible promoters are especially suitable ifgene expression is wanted to occur in a time specific manner. Examplesof such promoters are a salicylic acid inducible promoter (WO 95/19443),a tetracycline inducible promoter (Gatz et al., 1992 Plant J. 2:397-404)and an ethanol inducible promoter (WO 93/21334).

Also, suitable promoters responding to biotic or abiotic stressconditions are those such as the pathogen inducible PRPP1-gene promoter(Ward et al., 1993 Plant. Mol. Biol. 22:361-366), the heat induciblehsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold induciblealpha-amylase promoter from potato (WO 96/12814) or the wound-induciblepinII-promoter (EP 375091). For other examples of drought, cold, andsalt-inducible promoters, such as the RD29A promoter, seeYamaguchi-Shinozalei et al. (1993 Mol. Gen. Genet. 236:331-340).

Especially those promoters are preferred which confer gene expression inspecific tissues and organs, such as guard cells and the root haircells. Suitable promoters include the napin-gene promoter from rapeseed(U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumleinet al., 1991 Mol Gen Genet. 225(3):459-67), the oleosin-promoter fromArabidopsis (WO9845461), the phaseolin-promoter from Phaseolus vulgaris(U.S. Pat. No. 5,504,200), the Bce4-promoter from Brassica (WO9113980)or the legumin B4 promoter (LeB4; Baeumlein et al., 1992 Plant Journal,2(2):233-9) as well as promoters conferring seed specific expression inmonocot plants like maize, barley, wheat, rye, rice, etc. Suitablepromoters to note are the lpt2 or lpt1-gene promoter from barley (WO95/15389 and WO 95/23230) or those described in WO 99/16890 (promotersfrom the barley hordein-gene, rice glutelin gene, rice oryzin gene, riceprolamin gene, wheat gliadin gene, wheat glutelin gene, maize zein gene,oat glutelin gene, Sorghum kasirin-gene and rye secalin gene).

Also especially suited are promoters that confer plastid-specific geneexpression as plastids are the compartment where precursors and some endproducts of lipid biosynthesis are synthesized. Suitable promoters arethe viral RNA-polymerase promoter described in WO 95/16783 and WO97/06250 and the clpP-promoter from Arabidopsis described in WO99/46394.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to TFSRP mRNA. Regulatory sequences operatively linkedto a nucleic acid molecule cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types. For instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986 and Mol et al., 1990 FEBSLetters 268:427-430.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but they also apply to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aTFSRP can be expressed in bacterial cells such as C. glutamicum, insectcells, fungal cells or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells), algae, ciliates, plant cells, fungi or othermicroorganisms like C. glutamicum. Other suitable host cells are knownto those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation”, “transfection”, “conjugation” and“transduction” are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer and electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning. A Laboratory Manual. 2nd,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989) and other laboratory manuals such asMethods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols,ed: Gartland and Davey, Humana Press, Totowa, N.J. As biotic and abioticstress tolerance is a general trait wished to be inherited into a widevariety of plants like maize, wheat, rye, oat, triticale, rice, barley,soybean, peanut, cotton, rapeseed and canola, manihot, pepper, sunflowerand tagetes, solanaceous plants like potato, tobacco, eggplant, andtomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea),Salix species, trees (oil palm, coconut), perennial grasses and foragecrops, these crops plants are also preferred target plants for a geneticengineering as one further embodiment of the present invention.

In particular, the invention provides a method of producing a transgenicplant with a TFSRP coding nucleic acid, wherein expression of thenucleic acid in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a TFSRP nucleic acid, and (b) generating from the plant cella transgenic plant with a increased tolerance to environmental stress ascompared to a wild type variety of the plant. In preferred embodiments,the TFSRP is as described above. In preferred embodiments, the TFSRPcoding nucleic acid is as described above. The invention also provides amethod of increasing expression of a gene of interest within a host cellas compared to a wild type variety of the host cell, wherein the gene ofinterest is transcribed in response to a TFSRP, comprising: (a)transforming the host cell with an expression vector comprising a TFSRPcoding nucleic acid, and (b) expressing the TFSRP within the host cell,thereby increasing the expression of the gene transcribed in response tothe TFSRP as compared to a wild type variety of the host cell. Inpreferred embodiments, the TFSRP is as described above. In preferredembodiments, the TFSRP coding nucleic acid is as described above.

For such plant transformation, binary vectors such as pBinAR can be used(Höfgen and Willmitzer, 1990 Plant Science 66:221-230). Construction ofthe binary vectors can be performed by ligation of the cDNA in sense orantisense orientation into the T-DNA. 5-prime to the cDNA a plantpromoter activates transcription of the cDNA. A polyadenylation sequenceis located 3-prime to the cDNA. Tissue-specific expression can bearchived by using a tissue specific promoter. For example, seed-specificexpression can be archived by cloning the napin or LeB4 or USP promoter5-prime to the cDNA. Also, any other seed specific promoter element canbe used. For constitutive expression within the whole plant, the CaMV35S promoter can be used. The expressed protein can be targeted to acellular compartment using a signal peptide, for example for plastids,mitochondria or endoplasmic reticulum (Kermode, Crit. Rev. Plant Sci.,1996 4 (15):285-423). The signal peptide is cloned 5-prime in frame tothe cDNA to archive subcellular localization of the fusion protein.

Agrobacterium mediated plant transformation can be performed using forexample the GV3101(pMP90) (Koncz and Schell, 1986 Mol. Gen. Genet.204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain.Transformation can be performed by standard transformation techniques(Deblaere et al., 1994 Nucl. Acids. Res. 13:4777-4788). In oneembodiment, promoters that are responsive to abiotic stresses can beused with, such as the Arabidopsis promoter RD29A, the nucleic acidsequences disclosed herein. One skilled in the art will recognize thatthe promoter used should be operatively linked to the nucleic acid suchthat the promoter causes transcription of the nucleic acid which resultsin the synthesis of a mRNA which encodes a polypeptide. Alternatively,the RNA can be an antisense RNA for use in affecting subsequentexpression of the same or another gene or genes.

Agrobacterium mediated plant transformation can be performed usingstandard transformation and regeneration techniques (Gelvin, Stanton B.and Schilperoort, Robert A, Plant Molecular Biology Manual, 2ndEd.—Dordrecht: Kluwer Academic Publ., 1995.—in Sect., Ringbuc ZentraleSignatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R.; Thompson, JohnE., Methods in Plant Molecular Biology and Biotechnology, Boca Raton:CRC Press, 1993.-360 S.,ISBN 0-8493-5164-2). For example, rapeseed canbe transformed via cotyledon or hypocotyl transformation (Moloney etal., 1989 Plant cell Report 8:238-242; De Block et al., 1989 PlantPhysiol. 91:694-701). Use of antibiotica for Agrobacterium and plantselection depends on the binary vector and the Agrobacterium strain usedfor transformation. Rapeseed selection is normally performed usingkanamycin as selectable plant marker. Agrobacterium mediated genetransfer to flax can be performed using, for example, a techniquedescribed by Mlynarova et al., 1994 Plant Cell Report 13: 282-285.Additionally, transformation of soybean can be performed using forexample a technique described in EP 0424 047, U.S. Pat. No. 5,322,783(Pioneer Hi-Bred International) or in EP 0397 687, U.S. Pat. No.5,376,543, U.S. Pat. No. 5,169,770 (University Toledo).

Plant transformation using particle bombardment, Polyethylene Glycolmediated DNA uptake or via the Silicon Carbide Fiber technique is forexample described by Freeling and Walbot “The maize handbook” SpringerVerlag: New York (1993) ISBN 3-540-97826-7. A specific example of maizetransformation is found in U.S. Pat. No. 5,990,387 and a specificexample of wheat transformation can be found in WO 93/07256.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate or in plants thatconfer resistance towards a herbicide such as glyphosate or glufosinate.Nucleic acid molecules encoding a selectable marker can be introducedinto a host cell on the same vector as that encoding an TFSRP or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid molecule can be identified by, for example, drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

To create a homologous recombinant microorganism, a vector is preparedwhich contains at least a portion of a TFSRP gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the TFSRP gene. Preferably, this TFSRP gene is aPhyscomitrella patens TFSRP gene, but it can be a homologue from arelated plant or even from a mammalian, yeast, or insect source. In apreferred embodiment, the vector is designed such that, upon homologousrecombination, the endogenous TFSRP gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as aknock-out vector). Alternatively, the vector can be designed such that,upon homologous recombination, the endogenous TFSRP gene is mutated orotherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous TFSRP). To create a point mutation viahomologous recombination, DNA-RNA hybrids can be used in a techniqueknown as chimeraplasty (Cole-Strauss et al., 1999 Nucleic Acids Research27(5):1323-1330 and Kmiec, 1999 Gene therapy American Scientist.87(3):240-247). Homologous recombination procedures in Physcomitrellapatens are also well known in the art and are contemplated for useherein.

Whereas in the homologous recombination vector, the altered portion ofthe TFSRP gene is flanked at its 5′ and 3′ ends by additional nucleicacid molecule of the TFSRP gene to allow for homologous recombination tooccur between the exogenous TFSRP gene carried by the vector and anendogenous TFSRP gene in a microorganism or plant. The additionalflanking TFSRP nucleic acid molecule is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several hundreds of base pairs up to kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see e.g., Thomas, K. R.,and Capecchi, M. R., 1987 Cell 51:503 for a description of homologousrecombination vectors or Strepp et al., 1998 PNAS, 95 (8):4368-4373 forcDNA based recombination in Physcomitrella patens). The vector isintroduced into a microorganism or plant cell (e.g., via polyethyleneglycol mediated DNA) and cells in which the introduced TFSRP gene hashomologously recombined with the endogenous TFSRP gene are selected,using art-known techniques.

In another embodiment, recombinant microorganisms can be produced whichcontain selected systems which allow for regulated expression of theintroduced gene. For example, inclusion of a TFSRP gene on a vectorplacing it under control of the lac operon permits expression of theTFSRP gene only in the presence of IPTG. Such regulatory systems arewell known in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a TFSRP. Analternate method can be applied in addition in plants by the directtransfer of DNA into developing flowers via electroporation orAgrobacterium medium gene transfer. Accordingly, the invention furtherprovides methods for producing TFSRPs using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of invention (into which a recombinant expression vector encoding aTFSRP has been introduced, or into which genome has been introduced agene encoding a wild-type or altered TFSRP) in a suitable medium untilTFSRP is produced. In another embodiment, the method further comprisesisolating TFSRPs from the medium or the host cell.

Another aspect of the invention pertains to isolated TFSRPs, andbiologically active portions thereof. An “isolated” or “purified”protein or biologically active portion thereof is free of some of thecellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof TFSRP in which the protein is separated from some of the cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of TFSRP having less than about30% (by dry weight) of non-TFSRP (also referred to herein as a“contaminating protein”), more preferably less than about 20% ofnon-TFSRP, still more preferably less than about 10% of non-TFSRP, andmost preferably less than about 5% non-TFSRP. When the TFSRP orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation. The language “substantially free of chemical precursors orother chemicals” includes preparations of TFSRP in which the protein isseparated from chemical precursors or other chemicals that are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of TFSRP having less than about 30% (by dry weight) ofchemical precursors or non-TFSRP chemicals, more preferably less thanabout 20% chemical precursors or non-TFSRP chemicals, still morepreferably less than about 10% chemical precursors or non-TFSRPchemicals, and most preferably less than about 5% chemical precursors ornon-TFSRP chemicals. In preferred embodiments, isolated proteins orbiologically active portions thereof lack contaminating proteins fromthe same organism from which the TFSRP is derived. Typically, suchproteins are produced by recombinant expression of, for example, aPhyscomitrella patens TFSRP in plants other than Physcomitrella patensor microorganisms such as C. glutamicum, ciliates, algae or fungi.

An isolated TFSRP or a portion thereof of the invention can participatein a stress tolerance response in a plant, or more particularly canparticipate in the transcription of a protein involved in a stresstolerance response in a Physcomitrella patens plant, or has one or moreof the activities set forth in Table 1. In preferred embodiments, theprotein or portion thereof comprises an amino acid sequence which issufficiently homologous to an amino acid sequence encoded by a nucleicacid of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:22 such that the proteinor portion thereof maintains the ability to participate in themetabolism of compounds necessary for the construction of cellularmembranes in Physcomitrella patens, or in the transport of moleculesacross these membranes. The portion of the protein is preferably abiologically active portion as described herein. In another preferredembodiment, a TFSRP of the invention has an amino acid sequence of SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21 or SEQ ID NO:23. In yet another preferredembodiment, the TFSRP has an amino acid sequence which is encoded by anucleotide sequence which hybridizes, e.g., hybridizes under stringentconditions, to a nucleotide sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ IDNO:22. In still another preferred embodiment, the TFSRP has an aminoacid sequence which is at least about 50-60%, preferably at least about60-70%, more preferably at least about 70-80%, 80-90%, 90-95%, and evenmore preferably at least about 96%, 97%, 98%, 99% or more homologous toone of the amino acid sequences of SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ IDNO:23. The preferred TFSRPs of the present invention also preferablypossess at least one of the TFSRP activities described herein. Forexample, a preferred TFSRP of the present invention includes an aminoacid sequence encoded by a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, to a nucleotide sequence of SEQID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14 or SEQ ID NO:22, and which can participate canparticipate in a stress tolerance response in a plant, or moreparticularly can participate in the transcription of a protein involvedin a stress tolerance response in a Physcomitrella patens plant, orwhich has one or more of the activities set forth in Table 1.

In other embodiments, the TFSRP is substantially homologous to an aminoacid sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:23 and retains thefunctional activity of the protein of one of the sequences of SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21 or SEQ ID NO:23, yet differs in amino acid sequencedue to natural variation or mutagenesis, as described in detail above.Accordingly, in another embodiment, the TFSRP is a protein whichcomprises an amino acid sequence which is at least about 50-60%,preferably at least about 60-70%, and more preferably at least about70-80, 80-90, 90-95%, and most preferably at least about 96%, 97%, 98%,99% or more homologous to an entire amino acid sequence of SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21 or SEQ ID NO:23 and which has at least one of the TFSRPactivities described herein. In another embodiment, the inventionpertains to a full Physcomitrella patens protein which is substantiallyhomologous to an entire amino acid sequence encoded by a nucleic acid ofSEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14 or SEQ ID NO:22.

Biologically active portions of an TFSRP include peptides comprisingamino acid sequences derived from the amino acid sequence of an TFSRP,e.g., an amino acid sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ IDNO:23 or the amino acid sequence of a protein homologous to an TFSRP,which include fewer amino acids than a full length TFSRP or the fulllength protein which is homologous to an TFSRP, and exhibit at least oneactivity of an TFSRP. Typically, biologically active portions (peptides,e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37,38, 39, 40, 50, 100 or more amino acids in length) comprise a domain ormotif with at least one activity of a TFSRP. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the activities described herein. Preferably, the biologicallyactive portions of a TFSRP include one or more selected domains/motifsor portions thereof having biological activity.

TFSRPs are preferably produced by recombinant DNA techniques. Forexample, a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the TFSRP isexpressed in the host cell. The TFSRP can then be isolated from thecells by an appropriate purification scheme using standard proteinpurification techniques. Alternative to recombinant expression, a TFSRP,polypeptide, or peptide can be synthesized chemically using standardpeptide synthesis techniques. Moreover, native TFSRP can be isolatedfrom cells (e.g., Physcomitrella patens), for example using ananti-TFSRP antibody, which can be produced by standard techniquesutilizing a TFSRP or fragment thereof of this invention.

The invention also provides TFSRP chimeric or fusion proteins. As usedherein, a TFSRP “chimeric protein” or “fusion protein” comprises a TFSRPpolypeptide operatively linked to a non-TFSRP polypeptide. An “TFSRPpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a TFSRP, whereas a “non-TFSRP polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the TFSRP, e.g., a proteinwhich is different from the TFSRP and which is derived from the same ora different organism. Within the fusion protein, the term “operativelylinked” is intended to indicate that the TFSRP polypeptide and thenon-TFSRP polypeptide are fused to each other so that both sequencesfulfill the proposed function attributed to the sequence used. Thenon-TFSRP polypeptide can be fused to the N-terminus or C-terminus ofthe TFSRP polypeptide. For example, in one embodiment, the fusionprotein is a GST-TFSRP fusion protein in which the TFSRP sequences arefused to the C-terminus of the GST sequences. Such fusion proteins canfacilitate the purification of recombinant TFSRPs. In anotherembodiment, the fusion protein is a TFSRP containing a heterologoussignal sequence at its N-terminus. In certain host cells (e.g.,mammalian host cells), expression and/or secretion of a TFSRP can beincreased through use of a heterologous signal sequence.

Preferably, a TFSRP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). ATFSRP-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the TFSRP.

Homologues of the TFSRP can be generated by mutagenesis, e.g., discretepoint mutation or truncation of the TFSRP. As used herein, the term“homologue” refers to a variant form of the TFSRP which acts as anagonist or antagonist of the activity of the TFSRP. An agonist of theTFSRP can retain substantially the same, or a subset, of the biologicalactivities of the TFSRP. An antagonist of the TFSRP can inhibit one ormore of the activities of the naturally occurring form of the TFSRP, by,for example, competitively binding to a downstream or upstream member ofthe cell membrane component metabolic cascade which includes the TFSRP,or by binding to an TFSRP which mediates transport of compounds acrosssuch membranes, thereby preventing translocation from taking place.

In an alternative embodiment, homologues of the TFSRP can be identifiedby screening combinatorial libraries of mutants, e.g., truncationmutants, of the TFSRP for TFSRP agonist or antagonist activity. In oneembodiment, a variegated library of TFSRP variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of TFSRP variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential TFSRP sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of TFSRP sequences therein. There are avariety of methods which can be used to produce libraries of potentialTFSRP homologues from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene is then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential TFSRP sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A., 1983 Tetrahedron 39:3; Itakura et al., 1984 Annu. Rev. Biochem.53:323; Itakura et al., 1984 Science 198:1056; Ike et al., 1983 NucleicAcid Res. 11:477.

In addition, libraries of fragments of the TFSRP coding can be used togenerate a variegated population of TFSRP fragments for screening andsubsequent selection of homologues of a TFSRP. In one embodiment, alibrary of coding sequence fragments can be generated by treating adouble stranded PCR fragment of a TFSRP coding sequence with a nucleaseunder conditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the TFSRP.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of TFSRP homologues. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify TFSRP homologues (Arkin and Yourvan, 1992 PNAS 89:7811-7815;Delgrave et al., 1993 Protein Engineering 6(3):327-331). In anotherembodiment, cell based assays can be exploited to analyze a variegatedTFSRP library, using methods well known in the art. The presentinvention further provides a method of identifying a novel TFSRP,comprising (a) raising a specific antibody response to a TFSRP, orfragment thereof, as described above; (b) screening putative TFSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel TFSRP; and(c) analyzing the bound material in comparison to known TFSRP todetermine its novelty.

The nucleic acid molecules, proteins, protein homologues, fusionproteins, primers, vectors, and host cells described herein can be usedin one or more of the following methods: identification ofPhyscomitrella patens and related organisms; mapping of genomes oforganisms related to Physcomitrella patens; identification andlocalization of Physcomitrella patens sequences of interest;evolutionary studies; determination of TFSRP regions required forfunction; modulation of an TFSRP activity; modulation of the metabolismof one or more cell functions; modulation of the transmembrane transportof one or more compounds; and modulation of stress resistance.

The moss Physcomitrella patens represents one member of the mosses. Itis related to other mosses such as Ceratodon purpureus which is capableof growth in the absence of light. Mosses like Ceratodon andPhyscomitrella share a high degree of homology on the DNA sequence andpolypeptide level allowing the use of heterologous screening of DNAmolecules with probes evolving from other mosses or organisms, thusenabling the derivation of a consensus sequence suitable forheterologous screening or functional annotation and prediction of genefunctions in third species. The ability to identify such functions cantherefore have significant relevance, e.g., prediction of substratespecificity of enzymes. Further, these nucleic acid molecules may serveas reference points for the mapping of moss genomes, or of genomes ofrelated organisms.

The TFSRP nucleic acid molecules of the invention have a variety ofuses. Most importantly, the nucleic acid and amino acid sequences of thepresent invention can be used to transform plants, thereby inducingtolerance to stresses such as drought, high salinity and cold. Thepresent invention therefore provides a transgenic plant transformed by aTFSRP coding nucleic acid, wherein expression of the nucleic acidsequence in the plant results in increased tolerance to environmentalstress as compared to a wild type variety of the plant. The transgenicplant can be a monocot or a dicot. The invention further provides thatthe transgenic plant can be selected from maize, wheat, rye, oat,triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola,manihot, pepper, sunflower, tagetes, solanaceous plants, potato,tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao,tea, Salix species, oil palm, coconut, perennial grass and forage crops,for example. In particular, the present invention describes using theexpression of CABF-1 (SEQ ID NO:15), DBF-1 (SEQ ID NO:16), CBF-1 (SEQ IDNO:17), HDZ-1 (SEQ ID NO:18), ZF-1 (SEQ ID NO:19), LZ-1 (SEQ ID NO:20)and CABF-2(SEQ ID NO:21) to engineer drought-tolerant plants. Thisstrategy has herein been demonstrated for Arabidopsis thaliana,Rapeseed/Canola, soybeans, corn and wheat but its application is notrestricted to these plants. Accordingly, the invention provides atransgenic plant containing a TFSRP selected from 1) CABF-1; 2) CABF-2;3) DBF-1; 4) CBF-1; 5) HDZ-1; 6) ZF-1; 7) LZ-1 as defined above,including homologues, wherein the environmental stress is drought. Thisinvention also describes the principle of using over-expression of ZF-1(SEQ ID NO:19), CABF-2(SEQ ID NO:21) and LZ-1 (SEQ ID NO:20) to engineersalt-tolerant plants. Again, this strategy has herein been demonstratedfor Arabidopsis thaliana, Rapeseed/Canola, soybeans, corn and wheat butits application is not restricted to these plants. Accordingly, theinvention provides a transgenic plant containing the TFSRP selectedfrom 1) CABF-2; 2) ZF-1); and 3) LZ-1 as defined above, includinghomologues, wherein the environmental stress is salinity.

The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a TFSRP inthe plant. The invention provides that this method can be performed suchthat the stress tolerance is either increased or decreased.

Furthermore, this method can be used wherein the plant is eithertransgenic or not transgenic. In cases when the plant is transgenic, theplant can be transformed with a vector containing any of the abovedescribed TFSRP coding nucleic acids, or the plant can be transformedwith a promoter that directs expression of native TFSRP in the plant,for example. The invention provides that such a promoter can be tissuespecific. Furthermore, such a promoter can be developmentally regulated.Alternatively, non-transgenic plants can have native TFSRP expressionmodified by inducing a native promoter. Furthermore, the inventionprovides that TFSRP expression can be modified by administration of ananti-sense molecule that inhibits expression of TFSRP.

The expression of CABF-1 (SEQ ID NO:15), DBF-1 (SEQ ID NO:16), CBF-1(SEQID NO:17), HDZ-1(SEQ ID NO:18), ZF-1 (SEQ ID NO:19), LZ-1 (SEQ ID NO:20)and CABF-2(SEQ ID NO:21) in target plants can be accomplished by, but isnot limited to, one of the following examples: (a) constitutivepromoter, (b) stress-inducible promoter, (c) chemical-induced promoter,and (d) engineered promoter over-expression with for example zinc-fingerderived transcription factors (Greisman and Pabo, 1997 Science 275:657).The later case involves identification of the CABF-1 (SEQ ID NO:15),DBF-1 (SEQ ID NO:16), CBF-1 (SEQ ID NO:17), HDZ-1 (SEQ ID NO:18), ZF-1(SEQ ID NO:19), LZ-1 (SEQ ID NO:20) and CABF-2(SEQ ID NO:21) homologuesin the target plant as well as from its promoter. Zinc-finger-containingrecombinant transcription factors are engineered to specificallyinteract with the CABF-1 (SEQ ID NO:15), DBF-1 (SEQ ID NO:16), CBF-1(SEQ ID NO:17), HDZ-1(SEQ ID NO:18), ZF-1 (SEQ ID NO:19), LZ-1 (SEQ IDNO:20) and CABF-2(SEQ ID NO:21) homologue and transcription of thecorresponding gene is activated.

As shown herein and described more fully below, expression of the TFSRPs(CABF-1 (SEQ ID NO:15), DBF-1 (SEQ ID NO:16), CBF-1 (SEQ ID NO:17),HDZ-1(SEQ ID NO:18), ZF-1 (SEQ ID NO:19), LZ-1 (SEQ ID NO:20) and CABF-2(SEQ ID NO:21)) in Arabidopsis thaliana confers a high degree of droughttolerance to the plant. Additionally, several TFSRPs confer tolerance tohigh salt concentrations (ZF-1(SEQ ID NO:19), LZ-1 (SEQ ID NO:20) andCABF-2 (SEQ ID NO:21)) to this plant. Under drought stress conditions,CABF-1 over-expressing lines showed a survival rate of 89%, DBF-1over-expressing lines showed a survival rate of 80%, CBF-1over-expressing lines showed a survival rate of 100%; HDZ-1over-expressing lines showed a survival rate of 50%, ZF-1over-expressing lines showed a survival rate of 57%, LZ-1over-expressing lines showed a survival rate of 79%, and CABF-2over-expressing lines showed a survival rate of 50%. Under salt stressconditions, ZF-1 over-expressing lines showed a survival rate of 52%,CABF-2 over-expressing lines showed a survival rate of 56% and LZ-1over-expressing lines showed a survival rate of 48%. The untransformedcontrols showed a survival rate of 10%. It is noteworthy that theanalyses of these transgenic lines were performed with T1 plants.Therefore, the results will be better when a homozygous, strongexpresser is found. Further proof of involvement of these genes instress tolerance is given by the increase in the level of transcript inresponse to cold temperature treatment. The concentration of thetranscripts for CABF-1, CABF-2, and CBF-1 are all increased 2 fold overuntreated background following the treatment.

In addition to introducing the TFSRP nucleic acid sequences intotransgenic plants, these sequences can also be used to identify anorganism as being Physcomitrella patens or a close relative thereof.Also, they may be used to identify the presence of Physcomitrella patensor a relative thereof in a mixed population of microorganisms. Theinvention provides the nucleic acid sequences of a number ofPhyscomitrella patens genes; by probing the extracted genomic DNA of aculture of a unique or mixed population of microorganisms understringent conditions with a probe spanning a region of a Physcomitrellapatens gene which is unique to this organism, one can ascertain whetherthis organism is present.

Further, the nucleic acid and protein molecules of the invention mayserve as markers for specific regions of the genome. This has utilitynot only in the mapping of the genome, but also in functional studies ofPhyscomitrella patens proteins. For example, to identify the region ofthe genome to which a particular Physcomitrella patens DNA-bindingprotein binds, the Physcomitrella patens genome could be digested, andthe fragments incubated with the DNA-binding protein. Those which bindthe protein may be additionally probed with the nucleic acid moleculesof the invention, preferably with readily detectable labels; binding ofsuch a nucleic acid molecule to the genome fragment enables thelocalization of the fragment to the genome map of Physcomitrella patens,and, when performed multiple times with different enzymes, facilitates arapid determination of the nucleic acid sequence to which the proteinbinds. Further, the nucleic acid molecules of the invention may besufficiently homologous to the sequences of related species such thatthese nucleic acid molecules may serve as markers for the constructionof a genomic map in related mosses.

The TFSRP nucleic acid molecules of the invention are also useful forevolutionary and protein structural studies. The metabolic and transportprocesses in which the molecules of the invention participate areutilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the protein which are essential for the functioning of theenzyme. This type of determination is of value for protein engineeringstudies and may give an indication of what the protein can tolerate interms of mutagenesis without losing function.

Manipulation of the TFSRP nucleic acid molecules of the invention mayresult in the production of TFSRPs having functional differences fromthe wild-type TFSRPs. These proteins may be improved in efficiency oractivity, may be present in greater numbers in the cell than is usual,or may be decreased in efficiency or activity.

There are a number of mechanisms by which the alteration of a TFSRP ofthe invention may directly affect stress response and/or stresstolerance. In the case of plants expressing TFSRPs, increased transportcan lead to improved salt and/or solute partitioning within the planttissue and organs. By either increasing the number or the activity oftransporter molecules which export ionic molecules from the cell, it maybe possible to affect the salt tolerance of the cell.

The effect of the genetic modification in plants, C. glutamicum, fungi,algae, or ciliates on stress tolerance can be assessed by growing themodified microorganism or plant under less than suitable conditions andthen analyzing the growth characteristics and/or metabolism of theplant. Such analysis techniques are well known to one skilled in theart, and include dry weight, wet weight, protein synthesis, carbohydratesynthesis, lipid synthesis, evapotranspiration rates, general plantand/or crop yield, flowering, reproduction, seed setting, root growth,respiration rates, photosynthesis rates, etc. (Applications of HPLC inBiochemistry in: Laboratory Techniques in Biochemistry and MolecularBiology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3, Chapter III:Product recovery and purification, page 469-714, VCH: Weinheim; Belter,P. A. et al., 1988 Bioseparations: downstream processing forbiotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S.,1992 Recovery processes for biological materials, John Wiley and Sons;Shaeiwitz, J. A. and Henry, J. D., 1988 Biochemical separations, in:Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation andpurification techniques in biotechnology, Noyes Publications).

For example, yeast expression vectors comprising the nucleic acidsdisclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for fail or alteration oftheir tolerance to drought, salt, and temperature stress. Similarly,plant expression vectors comprising the nucleic acids disclosed herein,or fragments thereof, can be constructed and transformed into anappropriate plant cell such as Arabidopsis, soy, rape, maize, wheat,Medicago truncatula, etc., using standard protocols. The resultingtransgenic cells and/or plants derived therefrom can then be assayed forfail or alteration of their tolerance to drought, salt, and temperaturestress.

The engineering of one or more TFSRP genes of the invention may alsoresult in TFSRPs having altered activities which indirectly impact thestress response and/or stress tolerance of algae, plants, ciliates orfungi or other microorganisms like C. glutamicum. For example, thenormal biochemical processes of metabolism result in the production of avariety of products (e.g., hydrogen peroxide and other reactive oxygenspecies) which may actively interfere with these same metabolicprocesses (for example, peroxynitrite is known to nitrate tyrosine sidechains, thereby inactivating some enzymes having tyrosine in the activesite (Groves, J. T., 1999 Curr. Opin. Chem. Biol. 3(2):226-235). Whilethese products are typically excreted, cells can be genetically alteredto transport more products than is typical for a wild-type cell. Byoptimizing the activity of one or more TFSRPs of the invention which areinvolved in the export of specific molecules, such as salt molecules, itmay be possible to improve the stress tolerance of the cell.

Additionally, the sequences disclosed herein, or fragments thereof, canbe used to generate knockout mutations in the genomes of variousorganisms, such as bacteria, mammalian cells, yeast cells, and plantcells. (Girke, T., 1998 The Plant Journal 15:39-48). The resultantknockout cells can then be evaluated for their ability or capacity totolerate various stress conditions, their response to various stressconditions, and the effect on the phenotype and/or genotype of themutation. For other methods of gene inactivation include U.S. Pat. No.6,004,804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999Spliceosome-mediated RNA trans-splicing as a tool for gene therapyNature Biotechnology 17:246-252.

The aforementioned mutagenesis strategies for TFSRPs to result inincreased stress resistance are not meant to be limiting; variations onthese strategies will be readily apparent to one skilled in the art.Using such strategies, and incorporating the mechanisms disclosedherein, the nucleic acid and protein molecules of the invention may beutilized to generate algae, ciliates, plants, fungi or othermicroorganisms like C. glutamicum expressing mutated TFSRP nucleic acidand protein molecules such that the stress tolerance is improved.

The present invention also provides antibodies which specifically bindto a TFSRP-polypeptide, or a portion thereof, as encoded by a nucleicacid disclosed herein or as described herein. Antibodies can be made bymany well-known methods (See, e.g. Harlow and Lane, “Antibodies; ALaboratory Manual” Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1988)). Briefly, purified antigen can be injected into an animalin an amount and in intervals sufficient to elicit an immune response.Antibodies can either be purified directly, or spleen cells can beobtained from the animal. The cells can then fused with an immortal cellline and screened for antibody secretion. The antibodies can be used toscreen nucleic acid clone libraries for cells secreting the antigen.Those positive clones can then be sequenced. (See, for example, Kelly etal., 1992 Bio/Technology 10:163-167; Bebbington et al., 1992Bio/Technology 10:169-175).

The phrases “selectively binds” and “specifically binds” with thepolypeptide refers to a binding reaction which is determinative of thepresence of the protein in a heterogeneous population of proteins andother biologics. Thus, under designated immunoassay conditions, thespecified antibodies bound to a particular protein do not bind in asignificant amount to other proteins present in the sample. Selectivebinding to an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. A varietyof immunoassay formats may be used to select antibodies selectively bindwith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select antibodies selectively immunoreactive witha protein. See Harlow and Lane “Antibodies, A Laboratory Manual” ColdSpring Harbor Publications, New York, (1988), for a description ofimmunoassay formats and conditions that could be used to determineselective binding.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious hosts. A description of techniques for preparing such monoclonalantibodies may be found in Stites et al., editors, “Basic and ClinicalImmunology,” (Lange Medical Publications, Los Altos, Calif., FourthEdition) and references cited therein, and in Harlow and Lane(“Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, NewYork, 1988).

Throughout this application various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.Additionally, all references cited herein are hereby expresslyincorporated herein by reference.

EXAMPLES Example 1 Growth of Physcomitrella patens Cultures

For this study, plants of the species Physcomitrella patens (Hedw.) B.S. G. from the collection of the genetic studies section of theUniversity of Hamburg were used. They originate from the strain 16/14collected by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire(England), which was subcultured from a spore by Engel (1968, Am. J.Bot. 55:438-446). Proliferation of the plants was carried out by meansof spores and by means of regeneration of the gametophytes. Theprotonema developed from the haploid spore as a chloroplast-richchloronema and chloroplast-low caulonema, on which buds formed afterapproximately 12 days. These grew to give gametophores bearingantheridia and archegonia. After fertilization, the diploid sporophytewith a short seta and the spore capsule resulted, in which themeiospores matured.

Culturing was carried out in a climatic chamber at an air temperature of25° C. and light intensity of 55 micromol s⁻¹m⁻² (white light; PhilipsTL 65W/25 fluorescent tube) and a light/dark change of 16/8 hours. Themoss was either modified in liquid culture using Knop medium accordingto Reski and Abel (1985, Planta 165:354-358) or cultured on Knop solidmedium using 1% oxoid agar (Unipath, Basingstoke, England). Theprotonemas used for RNA and DNA isolation were cultured in aeratedliquid cultures. The protonemas were comminuted every 9 days andtransferred to fresh culture medium.

Example 2 Total DNA Isolation from Plants

The details for the isolation of total DNA relate to the working up ofone gram fresh weight of plant material. The materials used include thefollowing buffers: CTAB buffer: 2% (w/v)N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0;1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v)N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

The plant material was triturated under liquid nitrogen in a mortar togive a fine powder and transferred to 2 ml Eppendorf vessels. The frozenplant material was then covered with a layer of 1 ml of decompositionbuffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosine buffer, 20 μl ofβ-mercaptoethanol and 10 Ξl of proteinase K solution, 10 mg/ml) andincubated at 60° C. for one hour with continuous shaking. The homogenateobtained was distributed into two Eppendorf vessels (2 ml) and extractedtwice by shaking with the same volume of chloroform/isoamyl alcohol(24:1). For phase separation, centrifugation was carried out at 8000×gand room temperature for 15 minutes in each case. The DNA was thenprecipitated at −70° C. for 30 min using ice-cold isopropanol. Theprecipitated DNA was sedimented at 4° C. and 10,000 g for 30 minutes andresuspended in 180 μl of TE buffer (Sambrook et al., 1989, Cold SpringHarbor Laboratory Press: ISBN 0-87969-309-6). For further purification,the DNA was treated with NaCl (1.2 M final concentration) andprecipitated again at −70° C. for 30 minutes using twice the volume ofabsolute ethanol. After a washing step with 70% ethanol, the DNA wasdried and subsequently taken up in 50 μl of H₂O+RNAse (50 mg/ml finalconcentration). The DNA was dissolved overnight at 4° C. and the RNAsedigestion was subsequently carried out at 37° C. for 1 hour. Storage ofthe DNA took place at 4° C.

Example 3 Isolation of Total RNA and Poly-(A)+ RNA and cDNA LibraryConstruction from Physcomitrella Patens

For the investigation of transcripts, both total RNA and poly-(A)⁺ RNAwere isolated. The total RNA was obtained from wild-type 9 day oldprotonemata following the GTC-method (Reski et al., 1994 Mol. Gen.Genet. 244:352-359). The Poly(A)+ RNA was isolated using Dyna Beads®(Dynal, Oslo, Norway) following the instructions of the manufacturersprotocol. After determination of the concentration of the RNA or of thepoly(A)+ RNA, the RNA was precipitated by addition of 1/10 volumes of 3M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at −70° C.

For cDNA library construction, first strand synthesis was achieved usingMurine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany)and oligo-d(T)-primers, second strand synthesis by incubation with DNApolymerase I, Klenow enzyme and RNAseH digestion at 12° C. (2 hours),16° C. (1 hour) and 22° C. (1 hour). The reaction was stopped byincubation at 65° C. (10 minutes) and subsequently transferred to ice.Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche,Mannheim) at 37° C. (30 minutes). Nucleotides were removed byphenol/chloroform extraction and Sephadex G50 spin columns. EcoRIadapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends byT4-DNA-ligase (Roche, 12° C., overnight) and phosphorylated byincubation with polynucleotide kinase (Roche, 37° C., 30 minutes). Thismixture was subjected to separation on a low melting agarose gel. DNAmolecules larger than 300 base pairs were eluted from the gel, phenolextracted, concentrated on Elutip-D-columns (Schleicher and Schuell,Dassel, Germany) and were ligated to vector arms and packed into lambdaZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit(Stratagene, Amsterdam, Netherlands) using material and following theinstructions of the manufacturer.

Example 4 Sequencing and Function Annotation of Physcomitrella patensESTs

cDNA libraries as described in Example 2 were used for DNA sequencingaccording to standard methods, and in particular, by the chaintermination method using the ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany).Random Sequencing was carried out subsequent to preparative plasmidrecovery from cDNA libraries via in vivo mass excision,retransformation, and subsequent plating of DH10B on agar plates(material and protocol details from Stratagene, Amsterdam, Netherlands.Plasmid DNA was prepared from overnight grown E. coli cultures grown inLuria-Broth medium containing ampicillin (see Sambrook et al. 1989 ColdSpring Harbor Laboratory Press: ISBN 0-87969-309-6) on a Qiagene DNApreparation robot (Qiagen, Hilden) according to the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used:

5′-CAGGAAACAGCTATGACC-3′ SEQ ID NO:24 5′-CTAAAGGGAACAAAAGCTG-3′ SEQ IDNO:25 5′-TGTAAAACGACGGCCAGT-3′ SEQ ID NO:26

Sequences were processed and annotated using the software packageEST-MAX commercially provided by Bio-Max (Munich, Germany). The programincorporates practically all bioinformatics methods important forfunctional and structural characterization of protein sequences. Forreference the website at pedant.mips.biochem.mpg.de. The most importantalgorithms incorporated in EST-MAX are: FASTA: Very sensitive sequencedatabase searches with estimates of statistical significance; PearsonW.R., 1990 Rapid and sensitive sequence comparison with FASTP and FASTA.Methods Enzymol. 183:63-98; BLAST: Very sensitive sequence databasesearches with estimates of statistical significance. Altschul S. F.,Gish W., Miller W., Myers E. W., and Lipman D. J. Basic local alignmentsearch tool. Journal of Molecular Biology 215:403-10; PREDATOR:High-accuracy secondary structure prediction from single and multiplesequences. Frishman, D. and Argos, P., 1997 75% accuracy in proteinsecondary structure prediction. Proteins, 27:329-335; CLUSTALW: Multiplesequence alignment. Thompson, J. D., Higgins, D. G. and Gibson, T. J.(1994); CLUSTAL W: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, positions-specific gappenalties and weight matrix choice. Nucleic Acids Research,22:4673-4680; TMAP: Transmembrane region prediction from multiplyaligned sequences. Persson, B. and Argos, P., 1994 Prediction oftransmembrane segments in proteins utilizing multiple sequencealignments. J. Mol. Biol. 237:182-192; ALOM2: Transmembrane regionprediction from single sequences. Klein, P., Kanehisa, M., and DeLisi,C., 1984 Prediction of protein function from sequence properties: Adiscriminate analysis of a database. Biochim. Biophys. Acta 787:221-226.Version 2 by Dr. K. Nakai; PROSEARCH: Detection of PROSITE proteinsequence patterns. Kolakowski L. F. Jr., Leunissen J. A. M., Smith J.E., 1992 ProSearch: fast searching of protein sequences with regularexpression patterns related to protein structure and function.Biotechniques 13:919-921; BLIMPS: Similarity searches against a databaseof ungapped blocks. J. C. Wallace and Henikoff S., 1992; PATMAT: Asearching and extraction program for sequence, pattern and block queriesand databases, CABIOS 8:249-254. Written by Bill Alford.

Example 5 Identification of Physcomitrella patens ORF Corresponding toCABF-1, DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1 and CABF-2

The Physcomitrella patens partial cDNAs (ESTs) shown in Table 1 belowwere identified in the Physcomitrella patens EST sequencing programusing the program EST-MAX through BLAST analysis. (Tables 2-8 show someof these results). The Sequence Identification Numbers corresponding tothese ESTs are as follows: (SEQ ID NO:1), DBF-1 (SEQ ID NO:2), CBF-1(SEQ ID NO:3), HDZ-1 (SEQ ID NO:4), ZF-1 (SEQ ID NO:5), LZ-1 (SEQ IDNO:6) and CABF-2 (SEQ ID NO:7) These particular clones were chosen forfurther analyses since they encoded for transcription factors.

TABLE 1 Functional Category Putative Function Sequence Code ORF positionName Transcription DNA-binding protein s_pp00131077f  1-515 DBF-1 Factortranscription factor, c_pp004053131r 500-1  CABF-1 CCAAT-binding, chainA transcription factor s_pp004052093r  2-508 CABF-2 zinc finger proteinc_pp001074039r 1154-447  ZF-1 homeodomain leucine zipper c_pp001058012r364-750 HDZ-1 protein DNA-binding protein VBP1 s_pp013006061r  1-371LZ-1 transcriptional activator c_pp004032055r 183-998 CBF-1 CBF1

TABLE 2 Degree of amino acid identity and similarity of PpHDZ-1 andother homologous proteins (GCG Gap program was used: gap penalty: 10;gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # Q9LS31Q9LS33 Q43529 Q9XH37 Q9SP47 Protein name Homeobox Homeobox HomeoboxHomeodomain Homeodomain protein PPHB7 protein PPHB5 leucine zipperleucine protein zipper protein 57 Species Physcomitrella PhyscomitrellaLycopersicon Oryza sativa Glycine max patens (Moss) patens (Moss)esculentum (Rice) (Soybean) (Tomato) Identity % 71% 38% 30% 29% 30%Similarity % 72% 51% 40% 39% 36%

TABLE 3 Degree of amino acid identity and similarity of PpZF-1 and otherhomologous proteins (GCG Gap program was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Swiss-Prot # Q9SK53Q9ZTK7 Q9ZTK8 Q9XE47 O82431 Protein name Constans-like Constans-likeConstans-like Zinc finger Constans- B-box zinc protein 2 protein 1protein like 1 finger protein protein Species Arabidopsis ArabidopsisMalus Pinus radiata Raphanus thaliana thaliana domestica (Montereysativus (Mouse-ear (Mouse-ear (Apple) (Malus pine) (Radish) cress)cress) sylvestris) Identity % 40% 43% 42% 39% 41% Similarity % 50% 54%54% 49% 53%

TABLE 4 Degree of amino acid identity and similarity of PpCABF-1 andother homologous proteins (GCG Gap program was used: gap penalty: 10;gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # Q9ZQC3O23310 P25209 Q9LFI3 O23633 Protein name Putative CCAAT- CCAAT-Transcription Transcription CCAAT- binding binding factor NF-Y, factorbinding transcription transcription CCAAT- transcription factor subunitA factor subunit A binding-like factor protein Species ArabidopsisArabidopsis Zea mays Arabidopsis Arabidopsis thaliana thaliana (Maize)thaliana thaliana (Mouse-ear (Mouse-ear (Mouse-ear (Mouse-ear cress)cress) cress) cress) Identity % 47% 53% 49% 41% 46% Similarity % 58% 56%57% 53% 52%

TABLE 5 Degree of amino acid identity and similarity of PpDBF-1 andother homologous proteins (GCG Gap program was used: gap penalty: 10;gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # Q9ZUL5045609 Q9NPU9 Protein name Putative DNA- M03C11.8 Hypothetical bindingprotein protein 68.6 KDA protein Species Arabidopsis Caenorhabditis Homosapiens thaliana elegans (Human) (Mouse-ear cress) Identity % 47% 24%25% Similarity % 58% 35% 37%

TABLE 6 Degree of amino acid identity and similarity of PpCABF-2 andother homologous proteins (GCG Gap program was used: gap penalty: 10;gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # O23636Q9SNZ0 Q9SMP0 Q92869 O35088 Protein name Transcription Heme activatedTranscription Transcription Nuclear factor protein factor HAP5A factorNF-YC factor YC subunit Species Arabidopsis Arabidopsis Arabidopsis Homosapiens Mus thaliana thaliana thaliana (Human) musculus (Mouse-ear(Mouse-ear (Mouse-ear (Mouse) cress) cress) cress) Identity % 54% 40%42% 26% 25% Similarity % 62% 49% 49% 31% 30%

TABLE 7 Degree of amino acid identity and similarity of PpLZ-1 and otherhomologous proteins (GCG Gap program was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Swiss-Prot # Q9SQK1P43273 O24160 Q06979 Q41558 Protein name BZIP Transcription Leucinezipper OCS-element Transcription Transcription factor HBP- transcriptionbinding factor factor HBP- factor 1B factor TGA2.1 3.2 1B(C1) SpeciesNicotiana Arabidopsis Nicotiana Zea mays Triticum tabacum thalianatabacum (Maize) aestivum (Common (Mouse-ear (Common (Wheat) tobacco)cress) tobacco) Identity % 62% 73% 46% 46% 45% Similarity % 74% 61% 55%53% 53%

TABLE 8 Degree of amino acid identity and similarity of PpCBF-1 andother homologous proteins (GCG Gap program was used: gap penalty: 10;gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # orGenbank # Q9M210 BAA33435 Q9LU18 Q9ZQP3 Q9SUK8 Protein nameTranscription DREB1B Transcription Putative TINY Apetala2 factor-likefactor TINY- protein domain protein like protein TINY like proteinSpecies Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsisthaliana thaliana thaliana thaliana thaliana (Mouse-ear (Mouse-ear(Mouse-ear (Mouse-ear (Mouse-ear cress) cress) cress) cress) cress)Identity % 22% 21% 21% 20% 20% Similarity % 35% 32% 32% 30% 27%

Example 6 Cloning of the Full-Length Physcomitrella Patens cDNA encodingfor CABF-1, DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1, CABF-2

To isolate full-length CABF-1 (SEQ ID NO:8), CABF-2 (SEQ ID NO:14),CBF-1 (SEQ ID NO:10), PCR was performed as described below under thetitle “Full-length Amplification” using the original ESTs described inExample 4 as template since they were full-length (see Table 9 forprimers).

TABLE 9 Scheme and primers used for cloning of full-length clones Finalsites Isolation Gene in product Method Primers Race Primer RT-PCR DBF-1XmaI/HpaI 5′ RACE and RC056 RC279 RT-PCR for (SEQ ID NO:27) (SEQ IDNO:29) Full-length 5′GCGATCCTCAGCC 5′ATCCCGGGCGAT clone TGTCGATCCATT3′GGTGCGTTCGAGAT RC116 CGTAAGG3′ (SEQ ID NO:28) RC280 5′CCCTGAGGTATCG (SEQID NO:30) TTCCTGGTTCCC 5′GCGTTAACGAGC A3′ TTTCTCGCAGTGCC AGATAA3′ CABF-2XmaI/SacI PCR of RC031 original (SEQ ID NO:31) EST clone ATCCCGGGCTCTGCACCCCAGATGTCGC ATCCT RC032: (SEQ ID NO:32) CTGAGCTCTAATGC ATTCACTGTTGCTGCTGCT LZ-1 HpaI/EcoRV 5′ RACE and RC058 RC108 RT-PCR for (SEQ ID NO:33)(SEQ ID NO:34) Full-length 5′CCTGTAGGGCCAC 5′GAGTTAACGCAG cloneCCGGAGCTCACT3′ TGGTCACAACGCAG AGTACGC3′ RC109 (SEQ ID NO:35)5′GCGATATCGCTT CCATACCTGCGCCG AAGACTT3′ CBF01 XmaI/HpaI PCR of RC033original (SEQ ID NO:36) EST clone 5′GACCCGGGCCAT GTGATATGGCTTCA AAGTAT3′RC034 (SEQ ID NO:37) 5′GCGTTAACGACT CACTGAGAGTCATA ATGGTG3′ HDZ-1XmaI/HpaI 5′ RACE and RC047 RC321 RT-PCR for (SEQ ID NO:38) (SEQ IDNO:39) Full-length 5′CGTAGTCGCGCTC 5′ATCCCGGGCACG clone GAGCTGTTTGGT3′AGGGCAAGAGGGGA TAGAGAC3′ RC322 (SEQ ID NO:40) 5′GCGTTAACGCCGATGGTGCAACTTTG GTTGAC3′ ZF-1 XmaI/SacI 5′ RACE and RC063 RC122 RT-PCRfor (SEQ ID NO:41) (SEQ ID NO:42) Full-length 5′CCGTGTCCTCGGA5′ATCCCGGGAGGA clone GCATTCTGGCAT3′ GGGAGTTGGAATCT AGGAGAC3′ RC124 (SEQID NO:43) 5′GCGAGCTCGACC TTGCTCGATGGAGA CTCCAAT3′ CABF-1 XmaI/SacI PCRof RC019 original (SEQ ID NO:44) EST clone 5′ATCCCGGGAATA GGACGGATGGCCGACAGTTAC3′ RC020 (SEQ ID NO:45) 5′ATGAGCTCACTC TTACACTCCGCGGG GTTGGTT3′

To isolate the clones encoding for DBF-1 (SEQ ID NO:9), HDZ-1 (SEQ IDNO:11), ZF-1 (SEQ ID NO:12) and LZ-1 (SEQ ID NO:13) from Physcomitrellapatens, cDNA libraries were created with SMART RACE cDNA Amplificationkit (Clontech Laboratories) following manufacturer's instructions. TotalRNA isolated as described in Example 2 was used as the template. Thecultures were treated prior to RNA isolation as follows: Salt Stress: 2,6, 12, 24, 48 hours with 1-M NaCl-supplemented medium; Cold Stress: 4°C. for the same time points as for salt; Drought Stress: cultures wereincubated on dry filter paper for the same time points above. RNA wasthen pulled and used for isolation.

5′ RACE Protocol

The EST sequences DBF-1 (SEQ ID NO:2), HDZ-1 (SEQ ID NO:4), ZF-1 (SEQ IDNO:5) and LZ-1 (SEQ ID NO:6) identified from the database search asdescribed in Example 4 were used to design oligos for RACE (see Table9). The extended sequences for these genes were obtained by performingRapid Amplification of cDNA Ends polymerase chain reaction (RACE PCR)using the Advantage 2 PCR kit (Clontech Laboratories) and the SMART RACEcDNA amplification kit (Clontech Laboratories) using a Biometra T3Thermocycler following the manufacturer's instructions. The sequencesobtained from the RACE reactions corresponded to full-length codingregions of HDZ-1, ZF-1 and LZ-1 and were used to design oligos forfull-length cloning of the respective genes (see below full-lengthamplification). The RACE product of DBF-1 was not full length and a newRACE reaction was needed (see Table 9 for primers).

Full-Length Amplification

Full-length clones corresponding CABF-1 (SEQ ID NO:8), CBF-1 (SEQ IDNO:10) and CABF-2 (SEQ ID NO:14) were obtained by performing polymerasechain reaction (PCR) with gene-specific primers (see Table 9) and theoriginal EST as the template. The conditions for the reaction werestandard conditions with PWO DNA polymerase (Roche). PCR was performedaccording to standard conditions and to manufacturer's protocols(Sambrook et al., 1989 Molecular Cloning, A Laboratory Manual. 2ndEdition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.,Biometra T3 Thermocycler). The parameters for the reaction were: fiveminutes at 94° C. followed by five cycles of one minute at 94° C., oneminute at 50° C. and 1.5 minutes at 72° C. This was followed by twentyfive cycles of one minute at 94° C., one minute at 65° C. and 1.5minutes at 72° C.

Full-length clones corresponding to the DBF-1 (SEQ ID NO:9), HDZ-1 (SEQID NO:11), ZF-1 (SEQ ID NO:12) and LZ-1 (SEQ ID NO:13) genes wereisolated by repeating the RACE method but using the gene-specificprimers as given in Table 9.

The amplified fragments were extracted from agarose gel with a QIAquickGel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector(Invitrogen) following manufacturer's instructions. Recombinant vectorswere transformed into Top10 cells (Invitrogen) using standard conditions(Sambrook et al. 1989. Molecular Cloning, A Laboratory Manual. 2ndEdition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.).Transformed cells were selected for on LB agar containing 100 μg/mlcarbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside)and 0.8 mg IPTG (isopropylthio-β-D-galactoside) grown overnight at 37°C. White colonies were selected and used to inoculate 3 ml of liquid LBcontaining 100 μg/ml ampicillin and grown overnight at 37° C. PlasmidDNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Analyses of subsequent clones andrestriction mapping was performed according to standard molecularbiology techniques (Sambrook et al., 1989 Molecular Cloning, ALaboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press.Cold Spring Harbor, N.Y.).

Example 7 Engineering Stress-Tolerant Arabidopsis Plants byOver-Expressing the Genes CABF-1, DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1 andCABF-2

Binary Vector Construction: pGMSG

The pLMNC53 (Mankin, 2000, PhD thesis) vector was digested with HindIII(Roche) and blunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche)according to manufacturer's instructions. This fragment was extractedfrom agarose gel with a QIAquick Gel Extraction Kit (Qiagen) accordingto manufacturer's instructions. The purified fragment was then digestedwith EcoRI (Roche) according to manufacturer's instructions. Thisfragment was extracted from agarose gel with a QIAquick Gel ExtractionKit (Qiagen) according to manufacturer's instructions. The resulting 1.4kilobase fragment, the gentamycin cassette, included the nos promoter,aacCI gene and the g7 terminator.

The vector pBlueScript was digested with EcoRI and SmaI (Roche)according to manufacturer's instructions. The resulting fragment wasextracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen)according to manufacturer's instructions. The digested pBlueScriptvector and the gentamycin cassette fragments were ligated with T4 DNALigase (Roche) according to manufacturer's instructions, joining the tworespective EcoRI sites and joining the blunt-ended HindIII site with theSmaI site.

The recombinant vector (PGMBS) was transformed into Top10 cells(Invitrogen) using standard conditions. Transformed cells were selectedfor on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG(isopropylthio-β-D-galactoside), grown overnight at 37° C. Whitecolonies were selected and used to inoculate 3 ml of liquid LBcontaining 100 μg/ml ampicillin and grown overnight at 37° C. PlasmidDNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Analyses of subsequent clones andrestriction mapping was performed according to standard molecularbiology techniques (Sambrook et al. 1989. Molecular Cloning, ALaboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press.Cold Spring Harbor, N.Y.).

Both the pGMBS vector and plbxSuperGUS vector were digested with XbaIand KpnI (Roche) according to manufacturer's instructions, excising thegentamycin cassette from pGMBS and producing the backbone from theplbxSuperGUS vector. The resulting fragments were extracted from agarosegel with a QIAquick Gel Extraction Kit (Qiagen) according tomanufacturer's instructions. These two fragments were ligated with T4DNA ligase (Roche) according to manufacturer's instructions.

The resulting recombinant vector (pGMSG) was transformed into Top10cells (Invitrogen) using standard conditions. Transformed cells wereselected for on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG(isopropylthio-β-D-galactoside) and grown overnight at 37° C. Whitecolonies were selected and used to inoculate 3 ml of liquid LBcontaining 100 μg/ml ampicillin and grown overnight at 37° C. PlasmidDNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Analyses of subsequent clones andrestriction mapping was performed according to standard molecularbiology techniques (Sambrook et al., 1989 Molecular Cloning, ALaboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press.Cold Spring Harbor, N.Y.).

Subcloning of CABF-1, DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1 and CABF-2 intothe Binary Vector

The fragments containing the different Physcomitrella patenstranscription factors were subcloned from the recombinant PCR2.1 TOPOvectors by double digestion with restriction enzymes (see Table 10)according to manufacturer's instructions. The subsequence fragment wasexcised from agarose gel with a QIAquick Gel Extraction Kit (QIAgen)according to manufacturer's instructions and ligated into the binaryvector pGMSG, cleaved with appropriate enzymes (see Table 10) anddephosphorylated prior to ligation. The resulting recombinant pGMSGvector contained the corresponding transcription factor in the senseorientation under the control of the constitutive super promoter.

TABLE 10 Names of the various constructs of the Physcomitrella patenstranscription factors used for plant transformation Enzymes used togenerate gene Enzymes used to Binary Vector Gene fragment restrict pGMSGConstruct CABF-1 XmaI/SacI XmaI/SacI pBPSSH003 DBF-1 XmaI/HpaIXmaI/Ecl136 pBPSLVM009 CBF-1 XmaI/HpaI XmaI/Ecl136 pBPSSH002 HDZ-1XmaI/HpaI XmaI/Ecl136 pBPSLVM007 ZF-1 XmaI/SacI XmaI/SacI pBPSLVM008LZ-1 HpaI/EcoRV Ecl136 pBPSLVM012 CABF-2 XmaI/SacI XmaI/SacI pBPSMI003

Agrobacterium Transformation

The recombinant vectors were transformed into Agrobacterium tumefaciensC58Cl and PMP90 according to standard conditions (Hoefgen andWillmitzer, 1990).

Plant Transformation

Arabidopsis thaliana ecotype C24 were grown and transformed according tostandard conditions (Bechtold, 1993 Acad. Sci. Paris. 316:1194-1199;Bent et al., 1994 Science 265:1856-1860).

Screening of Transformed Plants

T1 seeds were sterilized according to standard protocols (Xiong et al.1999, Plant Molecular Biology Reporter 17: 159-170). Seeds were platedon ½ MS 0.6% agar supplemented with 1% sucrose, 150 μg/ml gentamycin(Sigma-Aldrich) and 2 μg/ml benomyl (Sigma-Aldrich). Seeds on plateswere vernalized for four days at 4° C. The seeds were germinated in aclimatic chamber at an air temperature of 22° C. and light intensity of40 micromol s⁻¹m⁻² (white light; Philips TL 65W/25 fluorescent tube) and16 hours light and 8 hours dark day length cycle. Transformed seedlingswere selected after 14 days and transferred to ½ MS 0.6% agar platessupplemented with 1% sucrose and allowed to recover for five-seven days.

Drought Tolerance Screening

T1 seedlings were transferred to dry, sterile filter paper in a petridish and allowed to desiccate for two hours at 80% RH (relativehumidity) in a Sanyo Growth Cabinet MLR-350H, micromole s⁻¹m⁻² (whitelight; Philips TL 65W/25 fluorescent tube). The RH was then decreased to60% and the seedlings were desiccated further for eight hours. Seedlingswere then removed and placed on ½ MS 0.6% agar plates supplemented with2 μg/ml benomyl and scored after five days.

The results of the drought tolerance screening in Arabidopsis thalianaplants over-expressing the TFSRP are shown in Table 11. It is noteworthythat these analyses were performed with T1 plants since the resultsshould be better when a homozygous, strong expresser is found.

TABLE 11 Summary of the drought stress tests Drought Stress Test GeneNumber of Total number of Percentage of Name survivors plants survivorsHDZ-1 7 14 50% ZF-1 25 45 53% CABF-1 8 9 89% DBF-1 4 5 80% CABF-2 3 650% LZ-1 11 14 79% CBF-1 9 9 100% Control 18 84 21%

Salt Tolerance Screening

Seedlings were transferred to filter paper soaked in ½ MS and placed on½ MS 0.6% agar supplemented with 2 μg/ml benomyl the night before thesalt tolerance screening. For the salt tolerance screening, the filterpaper with the seedlings was moved to stacks of sterile filter paper,soaked in 50 mM NaCl, in a petri dish. After two hours, the filter paperwith the seedlings was moved to stacks of sterile filter paper, soakedwith 200 mM NaCl, in a petri dish. After two hours, the filter paperwith the seedlings was moved to stacks of sterile filter paper, soakedin 600 mM NaCl, in a petri dish. After 10 hours, the seedlings weremoved to petri dishes containing ½ MS 0.6% agar supplemented with 2μg/ml benomyl. The seedlings were scored after 5 days.

The results of the salt tolerance screening in Arabidopsis thalianaplants over-expressing the TFSRPs are shown in Table 12. In particular,ZF-1 over-expressing Arabidopsis thaliana plants showed a 52% (12survivors from 23 stressed plants) survival rate; LZ-1, 48% (10 survivorfrom 21 stressed plants); CABF-2, 56% (5 survivors from 9 stressedplants); whereas the untransformed control a 9% (2 survivors from 23tested plants) survival rate. It is noteworthy that these analyses wereperformed with T1 plants, and therefore, the results should be betterwhen a homozygous, strong expresser is found.

TABLE 12 Summary of the salt stress tests Salt Stress Test Gene Numberof Total number of Percentage of Name survivors plants survivors ZF-1 1223 52% CABF-2 5 9 56% LZ-1 10 21 48% Control 2 23 9%

Freezing Tolerance Screening

Seedlings were moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2% sucrose and 2 μg/ml benomyl. After four days, theseedlings were incubated at 4° C. for 1 hour and then covered withshaved ice. The seedlings were then placed in an EnvironmentalSpecialist ES2000 Environmental Chamber and incubated for 3.5 hoursbeginning at −1.0° C. decreasing 1° C./'hour. The seedlings were thenincubated at −5.0° C. for 24 hours and then allowed to thaw at 5° C. for12 hours. The water was poured off and the seedlings were scored after 5days. The transgenic plants are then screened for their improved coldtolerance demonstrating that transgene expression confers coldtolerance.

Example 8 Detection of the CABF-1, DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1, andCABF-2 Transgenes in the Transgenic Arabidopsis Lines

One leaf from a wild type and a transgenic Arabidopsis plant washomogenized in 250 μl Hexadecyltrimethyl ammonium bromide (CTAB) buffer(2% CTAB, 1.4 M NaCl, 8 mM EDTA and 20 mM Tris pH 8.0) and 1 μlβ-mercaptoethanol. The samples were incubated at 60-65° C. for 30minutes and 250 μl of Chloroform was then added to each sample. Thesamples were vortexed for 3 minutes and centrifuged for 5 minutes at18,000×g. The supernatant was taken from each sample and 150 μlisopropanol was added. The samples were incubated at room temperaturefor 15 minutes, and centrifuged for 10 minutes at 18,000×g. Each pelletwas washed with 70% ethanol, dried, and resuspended in 20 μl TE. 4 μl ofabove suspension was used in a 20 μl PCR reaction using Taq DNApolymerase (Roche Molecular Biochemicals) according to themanufacturer's instructions. Binary vector plasmid containing each TFSRPgene was used as positive control, and the wild type C24 genomic DNA wasused as negative control in the PCR reactions. 10 μl PCR reaction wasanalyzed on 0.8% agarose-ethidium bromide gel.

The primers and reaction times used for amplification of each TFSRP geneare below. Notably, the transgenes were successfully amplified from theT1 transgenic lines, but not from the wild type C24. This resultindicates that the T1 transgenic plants contain at least one copy of thetransgenes. There was no indication of the existence of either identicalor very similar genes in the untransformed Arabidopsis thaliana controlwhich could be amplified by this method.

CABF-1

The primers used in the reactions were:

5′GAATAGATACGCTGACACGC3′ SEQ ID NO:465′ATGAGCTCACTCTTACACTCCGCGGGGTTGGTT3′ SEQ ID NO:47The PCR program was: 1 cycle of 1 minute at 94° C., 1 minute at 75° C.and 3 minutes at 72° C., followed by 14 cycles of the same cycle exceptthat the annealing temperature decreased 1° C. every cycle until 62° C.;and then 16 cycles of 1 minute at 94° C., 1 minute at 62° C. and 3minutes at 72° C. A 600-base pair fragment was generated from thepositive control and the transgenic plants.

HDZ-1

The primers used in the reactions were:

5′GAATAGATACGCTGACACGC3′ SEQ ID NO:465′GCGTTAACGCCGATGGTGCAACTTTGGTTGAC3′ SEQ ID NO:48The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.3-kb fragment was produced from the positive control and thetransgenic plants.

ZF-1

The primers used in the reactions were:

5′GAATAGATACGCTGACACGC3′ SEQ ID NO:465′GCGAGCTCGACCTTGCTCGATGGAGACTCCAAT3′ SEQ ID NO:49The PCR program was as following: 1 cycle of 1 minute at 94° C., 1minute at 75° C. and 3 minutes at 72° C., followed by 14 cycles of thesame cycle except that the annealing temperature decreased 1° C. everycycle until 62° C.; and then 16 cycles of 1 minute at 94° C., 1 minuteat 62° C. and 3 minutes at 72° C. A 1.3-kb fragment was generated fromthe positive control and the T1 transgenic plants.

CBF-1

The primers used in the reactions were:

5′GAATAGATACGCTGACACGC3′ SEQ ID NO:465′GCGTTAACGACTCACTGAGAGTCATAATGGTG3′ SEQ ID NO:50The PCR program was as following: 1 cycle of 1 minute at 94° C., 1minute at 75° C. and 3 minutes at 72° C., followed by 14 cycles of thesame cycle except that the annealing temperature decreased 1° C. everycycle until 62° C.; and then 16 cycles of 1 minute at 94° C., 1 minuteat 62° C. and 3 minutes at 72° C. A 1.1-kb fragment was generated fromthe positive control and the T1 transgenic plants.

DBF-1

The primers used in the reactions were:

5′CTAGTAACATAGATGACACC3′ SEQ ID NO:515′ATCCCGGGCGATGGTGCGTTCGAGATCGTAAGG3′ SEQ ID NO:52The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 2.9-kb fragment was produced from the positive control and thetransgenic plants.

CABF-2

The primers used in the reactions were:

5′GAATAGATACGCTGACACGC3′ SEQ ID NO:535′CTGAGCTCTAATGCATTCACTGTTGCTGCTGCT3′ SEQ ID NO:54The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. An 800-bp fragment was produced from the positive control and thetransgenic plants.

LZ-1

The primers used in the reactions were:

5′GAATAGATACGCTGACACGC3′ SEQ ID NO:535′GCGATATCGCTTCCATACCTGCGCCGAAGACTT3′ SEQ ID NO:55The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C. and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.8-kb fragment was produced from the positive control and thetransgenic plants.

Example 9 Detection of the CABF-1, DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1, andCABF-2 Transgene mRNA in Transgenic Arabidopsis Lines

Transgene expression was detected using RT-PCR. Total RNA was isolatedfrom stress-treated plants using a procedure adapted from (Verwoerd etal., 1989 NAR 17:2362). Leaf samples (50-100 mg) were collected andground to a fine powder in liquid nitrogen. Ground tissue wasresuspended in 500 μl of an 80° C., 1:1 mixture, of phenol to extractionbuffer (100 mM LiCl, 100 mM Tris pH8, 10 mM EDTA, 1% SDS), followed bybrief vortexing to mix. After the addition of 250 μl of chloroform, eachsample was vortexed briefly. Samples were then centrifuged for 5 minutesat 12,000×g. The upper aqueous phase was removed to a fresh eppendorftube. RNA was precipitated by adding 1/10^(th) volume 3M sodium acetateand 2 volumes 95% ethanol. Samples were mixed by inversion and placed onice for 30 minutes. RNA was pelleted by centrifugation at 12,000×g for10 minutes. The supernatant was removed and pellets briefly air-dried.RNA sample pellets were resuspended in 10 μl DEPC treated water. Toremove contaminating DNA from the samples, each was treated withRNAse-free DNAse (Roche) according to the manufacturer'srecommendations. cDNA was synthesized from total RNA using the 1^(st)Strand cDNA synthesis kit (Boehringer Mannheim) following manufacturer'srecommendations. PCR amplification of a gene-specific fragment from thesynthesized cDNA was performed using Taq DNA polymerase (Roche) andgene-specific primers (see Table 13 for primers) in the followingreaction: 1×PCR buffer, 1.5 mM MgCl₂, 0.2 μM each primer, 0.2 μM dNTPs,1 unit polymerase, 5 μl cDNA from synthesis reaction. Amplification wasperformed under the following conditions: Denaturation, 95° C., 1minute; annealing, 62° C., 30 seconds; extension, 72° C., 1 minute, 35cycles; extension, 72° C., 5 minutes; hold, 4° C., forever. PCR productswere run on a 1% agarose gel, stained with ethidium bromide, andvisualized under UV light using the Quantity-One gel documentationsystem (Bio-Rad).

Expression of the transgenes was detected in the T1 transgenic line.These results indicated that the transgenes are expressed in thetransgenic lines and strongly suggested that their gene product improvedplant stress tolerance in the transgenic lines. In agreement with theprevious statement, no expression of identical or very similarendogenous genes could be detected by this method. These results are inagreement with the data from Example 7.

TABLE 13 Primers used for the amplification of respec- tive transgenemRNA in PCR using RNA isolated from transgenic Arabidopsisthaliana plants as template Gene 5′ primer 3′ primer DBF-1 RC876 RC877(SEQ ID NO:56) (SEQ ID NO:57) 5′GGAGACGGTATCACACCATC 5′TGCACAGACATCTGCCTGAAGA3′ GGCTCACA3′ CABF-2 RC974 RC975 (SEQ ID NO:58) (SEQ ID NO:60)5′GATGATCGCAGCCGAAGCTC 5′GGCAGTCTGTGGAGGCT CAGTG3′ GATACATCA3′ RC976:RC977: (SEQ ID NO:59) (SEQ ID NO:61) 5′GGGTGTGCCATGGACTGGT5′CCTGATCCTGTGACCCC GTTCCAG3′ TTTTGCCA3′ LZ-1 RC978 RC979 (SEQ ID NO:62)(SEQ ID NO:64) 5′GACATGGACGGTGATGCGA 5′GCATACTCCAGGTCAAA AGTTGG3′TGCAGCAGC3′ RC980: RC981: (SEQ ID NO:63) (SEQ ID NO:65)5′CGGCAACAGCAGGGTCTAT 5′GGGTCGGCAGCCTCCAA ACCTTGG3′ TCCATACA3′ CBF-1RC880 RC881 (SEQ ID NO:66) (SEQ ID NO:67) 5′GGCAGGGAATCTACGCATC5′CGACGAGATTCTCTGCA GCTTTG3′ ACATCTGAG3′ HDZ-1 RC982 RC983 (SEQ IDNO:68) (SEQ ID NO:69) 5′GGAGCTTGGACTGCGACCTC 5′GGTGTGGCTCGTGCGAGGTCAAG3′ GGCTATCAG3′ RC984: RC985: (SEQ ID NO:70) (SEQ ID NO:71)5′GTCATCGAGGAATCGCACA 5′GGTTGACGTTGGATTGC ACTCCT3′ ACATGGTGG3′ ZF-1RC874 RC875 (SEQ ID NO:72) (SEQ ID NO:73) 5′TGGATGTGCGAAGTGTGCG5′GCGCTGCCTCTGATAAT AGGTTG3′ AGAGTTGG3′ CABF-1 RC938 RC939 (SEQ IDNO:74) (SEQ ID NO:75) 5′GTGCAGGAGTGCGTATCCG 5′CGTACGGCTGTTGCATCAGTTCATC3′ ATCTGCATCG3′

Example 10 Engineering Stress-Tolerant Soybean Plants by Over-Expressingthe CABF-1, DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1 and CABF-2 Gene

The constructs pBPSLVM111, pBPSLVM149, pBPSLVM157, pBPSLVM39, pBPSLVM12,pBPSLVM19, pBPSLVM69 are used to transform soybean as described below.

Seeds of soybean are surface sterilized with 70% ethanol for 4 minutesat room temperature with continuous shaking, followed by 20% (v/v)Clorox supplemented with 0.05% (v/v) Tween for 20 minutes withcontinuous shaking. Then, the seeds are rinsed 4 times with distilledwater and placed on moistened sterile filter paper in a Petri dish atroom temperature for 6 to 39 hours. The seed coats are peeled off, andcotyledons are detached from the embryo axis. The embryo axis isexamined to make sure that the meristematic region is not damaged. Theexcised embryo axes are collected in a half-open sterile Petri dish andair-dried to a moisture content less than 20% (fresh weight) in a sealedPetri dish until further use.

Agrobacterium tumefaciens culture is prepared from a single colony in LBsolid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin,50 mg/l kanamycin) followed by growth of the single colony in liquid LBmedium to an optical density at 600 nm of 0.8. Then, the bacteriaculture is pelleted at 7000 rpm for 7 minutes at room temperature, andresuspended in MS (Murashige and Skoog, 1962) medium supplemented with100 μM acetosyringone. Bacteria cultures are incubated in thispre-induction medium for 2 hours at room temperature before use. Theaxis of soybean zygotic seed embryos at approximately 15% moisturecontent are imbibed for 2 hours at room temperature with the pre-inducedAgrobacterium suspension culture. The embryos are removed from theimbibition culture and are transferred to Petri dishes containing solidMS medium supplemented with 2% sucrose and incubated for 2 days, in thedark at room temperature. Alternatively, the embryos are placed on topof moistened (liquid MS medium) sterile filter paper in a Petri dish andincubated under the same conditions described above. After this period,the embryos are transferred to either solid or liquid MS mediumsupplemented with 500 mg/L carbenicillin or 300 mg/L cefotaxime to killthe agrobacteria. The liquid medium is used to moisten the sterilefilter paper. The embryos are incubated during 4 weeks at 25° C., under150 μmol m⁻²sec⁻¹ and 12 hours photoperiod. Once the seedlings produceroots, they are transferred to sterile metromix soil. The medium of thein vitro plants is washed off before transferring the plants to soil.The plants are kept under a plastic cover for 1 week to favor theacclimatization process. Then the plants are transferred to a growthroom where they are incubated at 25° C., under 150 μmol m⁻²sec⁻¹ lightintensity and 12 hours photoperiod for about 80 days.

The transgenic plants are then screened for their improved drought, saltand/or cold tolerance according to the screening method described inExample 7 to demonstrate that transgene expression confers stresstolerance.

Example 11 Engineering Stress-Tolerant Rapeseed/Canola Plants byOver-Expressing the CABF-1; DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1 and CABF-2Gene

The constructs pBPSLVM111, pBPSLVM149, pBPSLVM157, pBPSLVM39, pBPSLVM12,pBPSLVM19, pBPSLVM69 are used to transform rapeseed/canola as describedbelow.

The method of plant transformation described herein is also applicableto Brassica and other crops. Seeds of canola are surface sterilized with70% ethanol for 4 minutes at room temperature with continuous shaking,followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20minutes, at room temperature with continuous shaking. Then, the seedsare rinsed 4 times with distilled water and placed on moistened sterilefilter paper in a Petri dish at room temperature for 18 hours. Then theseed coats are removed and the seeds are air dried overnight in ahalf-open sterile Petri dish. During this period, the seeds lose approx.85% of its water content. The seeds are then stored at room temperaturein a sealed Petri dish until further use. DNA constructs and embryoimbibition are as described in Example 10. Samples of the primarytransgenic plants (T0) are analyzed by PCR to confirm the presence ofT-DNA. These results are confirmed by Southern hybridization in whichDNA is electrophoresed on a 1% agarose gel and transferred to apositively charged nylon membrane (Roche Diagnostics). The PCR DIG ProbeSynthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

The transgenic plants are then screened for their improved stresstolerance according to the screening method described in Example 7demonstrating that transgene expression confers drought tolerance.

Example 12 Engineering Stress-Tolerant Corn Plants by Over-Expressingthe CABF-1; DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1 and CABF-2 Gene

The constructs pBPSLVM111, pBPSLVM149, pBPSLVM157, pBPSLVM39, pBPSLVM12,pBPSLVM19, pBPSLVM69 are used to transform corn as described below.

Transformation of maize (Zea Mays L.) is performed with the methoddescribed by Ishida et al., 1996 Nature Biotech 14745-50. Immatureembryos are co-cultivated with Agrobacterium tumefaciens that carry“super binary” vectors, and transgenic plants are recovered throughorganogenesis. This procedure provides a transformation efficiency ofbetween 2.5% and 20%. The transgenic plants are then screened for theirimproved drought, salt and/or cold tolerance according to the screeningmethod described in Example 7 demonstrating that transgene expressionconfers stress tolerance.

Example 13 Engineering Stress-Tolerant Wheat Plants by Over-Expressingthe CABF-1, DBF-1, CBF-1, HDZ-1, ZF-1, LZ-1 and CABF-2 Gene

The constructs pBPSLVM111, pBPSLVM149, pBPSLVM157, pBPSLVM39, pBPSLVM12,pBPSLVM19, pBPSLVM69 are used to transform wheat as described below.

Transformation of wheat is performed with the method described by Ishidaet al. 1996. Nature Biotech 14745-50. Immature embryos are co-cultivatedwith Agrobacterium tumefaciens that carry “super binary” vectors, andtransgenic plants are recovered through organogenesis. This procedureprovides a transformation efficiency between 2.5% and 20%. Thetransgenic plants are then screened for their improved stress toleranceaccording to the screening method described in Example 7 demonstratingthat transgene expression confers drought tolerance.

Example 14 Monitoring Changes in mRNA Concentration of CABF-1, CABF-2and CBF-1 in Physcomitrella Patens Cultures Cold Treated DNA MicroarraySlide Preparation

PCR amplification was performed in 96 well plates from selectedPhyscomitrella patens ESTs cloned in the pBluescript vector. The PCRbuffer set (Boehringer Mannheim) was employed for PCR reaction. Each PCRreaction mixture contains 10 μl of PCR Buffer without MgCl₂, 10 μl ofMgSO₄, 3 μl of SK-Fwd primer (MWG-Biotech, Sequence:5′-CGCCAAGCGCGCAATTAACCCTCACT-3′, SEQ ID NO:76), 3 μl SK-Rev primer(MWG-Biotech, Sequence: 5′GCGTAATACGACTCACTATAGGG CGA-3′, SEQ ID NO:77),2 μl dNTP, 1 μl Taq DNA polymerase (Roche), 72 μl water and 1 μl DNAtemplate. After denaturing at 95° C. for three minutes, the PCRreactions were performed with 35 cycles of three consecutive stepsincluding denaturing at 95° C. for 45 seconds, annealing at 63° C. for45 seconds, and elongation at 72° C. for 60 seconds. The last elongationwas 72° C. for 10 minutes. The PCR products were then purified withQIAquick PCR purification kit (Qiagen, Inc.), eluted with water and theDNA concentration measured at 260 nm in a spectrophotometer.

2 to 5 μg of each PCR product were dried down and dissolved in 50 μl ofDMSO. The PCR products were then formatted from 96 well plates to 384well plates for printing. Microarray GenIII arrayer (Molecular Dynamics)was employed to print the PCR products to microarray slides (MolecularDynamics) with the format recommended by the manufacturer. The printedspots were about 290 μm in diameter and were spaced about 320 μm fromcenter to center. After printing, the slide was left in the dust freechamber for one hour to dry out. UV cross-link was performed with 600μJ/mm. The cross-linked slides were ready for hybridization and werestored in dark and dry chambers.

Microarray Probe Synthesis

Total RNA was extracted from cold-treated Physcomitrella patens cultures(12 hours at 4° C. in the dark) following the RNA extraction methoddescribed in Ausubel et al. (1987 Curr. Prot. in Mol. Biol. J. Wiley andSons, New York). Oligotex mRNA midi kit (Qiagen Inc.) was applied toisolate mRNA from total RNA with an approach combining both batch andstandard protocol recommended by the manufacturer. After binding thetotal RNA with Oligotex, the sample was centrifuged at 14000×g toseparate the Oligotex:mRNA with the liquid phase instead of runningthrough a column. After four washes with OW2 buffer as described inbatch protocol, the Oligotex:mRNA was resuspended in 400 μl OW2 and thencollected by the column as the standard protocol. The mRNA was elutedfollowing standard protocol.

Cy3 and Cy5 labeled cDNA probes were synthesized from mRNA withSuperscript Choice System for cDNA synthesis (Gibco BRL). Botholigo-(dT)₂₅ primer (Genosys Biotechnologies) and Nonamer primer(Amersham Pharmacia Biotech) were mixed with mRNA to reach a totalvolume of 20 μl. The mixture was first heated at 70° C. for 10 minutesand then left at room temperature for 15 minutes before transferring toice. Once the sample is on ice, add 8 μl First Strand Synthesis Buffer,4 μl 0.1M DTT, 2 μl dNTP (Amersham Pharmacia Biotech), 2 μl Cy3- orCy5-dCTP (Amersham Pharmacia Biotech), 2 μl RNAse Inhibitor (Gibco BRL)and 2 μl SuperScript II Reverse Transcriptase. The first strandsynthesis was performed at 42° C. for 8 hours and the mixture was thenheated at 94° C. for three minutes after the reaction.

After the first strand synthesis, 4 μl of 2.5M sodium hydroxide wasadded to the reaction and the mixture was incubated at 37° C. for tenminutes. 20 μl of 2M MOPS (pH 5.0) and 500 μl of PB buffer (Qiagen Inc.)were then added to each reaction. The probe was then purified by theQIAquick PCR Purification Kit (Qiagen Inc.) with the protocol providedby the manufacturer.

cDNA Microarray Hybridization and Washes

The purified Cy3- and Cy5-labeled probes were mixed and vacuum died togive a final volume of 9 μl. 9 μl Microarray Hybridization Solution(Amersham Pharmacia Biotech) and 18 μl Formamide (Sigma) were then addedto the cDNA probes to give a final volume of 36 μl. The mixture wasapplied to the printed microarray slide that was then covered with aclean dust-free cover slide with no air trapped. The hybridization wasperformed in a hybridization chamber at 42° C. for 16 to 20 hours. Afterthe hybridization, the slides were washed two times with 0.5×SSC, 0.2%SDS at room temperature for 5 minutes and 15 minutes. Two times ofstringent washes were performed with 0.25×SSC, 0.1% SDS at 55° C. for 10and 30 minutes respectively. After the washes, the slides were brieflyrinsed with Millipore water and dried under compressed nitrogen.

Scanning, Microarray Data Analysis

The cDNA microarrays were scanned using the microarray GenIII Scanner(Molecular Dynamics) equipped with two laser channels. The scannedimages were firstly viewed and adjusted in ImageQuant software(Molecular Dynamics) and then analyzed by ArrayVision software(Molecular Dynamics). The signal intensity for each spot was extractedby ArrayVision software (Molecular Dynamics) and transferred to Excel(Microsoft). The data obtained was normalized by dividing the differenceof the intensity value and background and the difference of the controlvalue and background. The ratio was then obtained by dividing thenormalized data.

Example 15 Identification of Homologous and Heterologous Genes

Gene sequences can be used to identify homologous or heterologous genesfrom cDNA or genomic libraries. Homologous genes (e.g. full-length cDNAclones) can be isolated via nucleic acid hybridization using for examplecDNA libraries. Depending on the abundance of the gene of interest,100,000 up to 1,000,000 recombinant bacteriophages are plated andtransferred to nylon membranes. After denaturation with alkali, DNA isimmobilized on the membrane by e.g. UV cross linking. Hybridization iscarried out at high stringency conditions. In aqueous solutionhybridization and washing is performed at an ionic strength of 1 M NaCland a temperature of 68° C. Hybridization probes are generated by e.g.radioactive (³²P) nick transcription labeling (High Prime, Roche,Mannheim, Germany). Signals are detected by autoradiography.

Partially homologous or heterologous genes that are related but notidentical can be identified in a manner analogous to the above-describedprocedure using low stringency hybridization and washing conditions. Foraqueous hybridization, the ionic strength is normally kept at 1 M NaClwhile the temperature is progressively lowered from 68 to 42° C.

Isolation of gene sequences with homologies (or sequenceidentity/similarity) only in a distinct domain of (for example 10-20amino acids) can be carried out by using synthetic radio labeledoligonucleotide probes. Radio labeled oligonucleotides are prepared byphosphorylation of the 5-prime end of two complementary oligonucleotideswith T4 polynucleotide kinase. The complementary oligonucleotides areannealed and ligated to form concatemers. The double strandedconcatemers are than radiolabeled by, for example, nick transcription.Hybridization is normally performed at low stringency conditions usinghigh oligonucleotide concentrations.

Oligonucleotide hybridization solution:

6×SSC

0.01 M sodium phosphate

1 mM EDTA (pH 8) 0.5% SDS

100 μg/ml denatured salmon sperm DNA0.1% nonfat dried milk

During hybridization, temperature is lowered stepwise to 5-10° C. belowthe estimated oligonucleotide Tm or down to room temperature followed bywashing steps and autoradiography. Washing is performed with lowstringency such as 3 washing steps using 4×SSC. Further details aredescribed by Sambrook, J. et al., 1989, “Molecular Cloning: A LaboratoryManual”, Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al.,1994 “Current Protocols in Molecular Biology”, John Wiley & Sons.

Example 16 Identification of Homologous Genes by Screening ExpressionLibraries with Antibodies

cDNA clones can be used to produce recombinant protein for example in E.coli (e.g. Qiagen QIAexpress pQE system). Recombinant proteins are thennormally affinity purified via Ni-NTA affinity chromatography (Qiagen).Recombinant proteins are then used to produce specific antibodies forexample by using standard techniques for rabbit immunization. Antibodiesare affinity purified using a Ni-NTA column saturated with therecombinant antigen as described by Gu et al., 1994 BioTechniques17:257-262. The antibody can than be used to screen expression cDNAlibraries to identify homologous or heterologous genes via animmunological screening (Sambrook, J. et al., 1989, “Molecular Cloning:A Laboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al., 1994 “Current Protocols in Molecular Biology”, John Wiley &Sons).

Example 17 In Vivo Mutagenesis

In vivo mutagenesis of microorganisms can be performed by passage ofplasmid (or other vector) DNA through E. coli or other microorganisms(e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) whichare impaired in their capabilities to maintain the integrity of theirgenetic information. Typical mutator strains have mutations in the genesfor the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; forreference, see Rupp, W. D., 1996 DNA repair mechanisms, in: Escherichiacoli and Salmonella, p. 2277-2294, A S M: Washington.) Such strains arewell known to those skilled in the art. The use of such strains isillustrated, for example, in Greener, A. and Callahan, M., 1994Strategies 7:32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theexemplification of this document.

Example 18 In Vitro Analysis of the Function of Physcomitrella Genes inTransgenic Organisms

The determination of activities and kinetic parameters of enzymes iswell established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one skilled inthe art. Overviews about enzymes in general, as well as specific detailsconcerning structure, kinetics, principles, methods, applications andexamples for the determination of many enzyme activities may be found,for example, in the following references: Dixon, M., and Webb, E. C.,(1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure andMechanism. Freeman: New York; Walsh, (1979) Enzymatic ReactionMechanisms. Freeman: San Francisco; Price, N. C., Stevens, L. (1982)Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D.,ed. (1983) The Enzymes, 3^(rd) ed. Academic Press: New York; Bisswanger,H., (1994) Enzymkinetik, 2^(nd) ed. VCH: Weinheim (ISBN 3527300325);Bergmeyer, H. U., Bergmeyer, J., Graβl, M., eds. (1983-1986) Methods ofEnzymatic Analysis, 3^(rd) ed., vol. I-XII, Verlag Chemie: Weinheim; andUllmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes.V C H: Weinheim, p. 352-363.

The activity of proteins which bind to DNA can be measured by severalwell-established methods, such as DNA band-shift assays (also called gelretardation assays). The effect of such proteins on the expression ofother molecules can be measured using reporter gene assays (such as thatdescribed in Kolmar, H. et al., 1995 EMBO J. 14:3895-3904 and referencescited therein). Reporter gene test systems are well known andestablished for applications in both pro- and eukaryotic cells, usingenzymes such as β-galactosidase, green fluorescent protein, and severalothers.

The determination of activity of membrane-transport proteins can beperformed according to techniques such as those described in Gennis, R.B. Pores, Channels and Transporters, in Biomembranes, MolecularStructure and Function, pp. 85-137, 199-234 and 270-322, Springer:Heidelberg (1989).

Example 19 Purification of the Desired Product from TransformedOrganisms

Recovery of the desired product from plant material (i.e.,Physcomitrella patents or Arabidopsis thaliana), fungi, algae, ciliates,C. glutamicum cells, or other bacterial cells transformed with thenucleic acid sequences described herein, or the supernatant of theabove-described cultures can be performed by various methods well knownin the art. If the desired product is not secreted from the cells, canbe harvested from the culture by low-speed centrifugation, the cells canbe lysed by standard techniques, such as mechanical force orsonification. Organs of plants can be separated mechanically from othertissue or organs. Following homogenization cellular debris is removed bycentrifugation, and the supernatant fraction containing the solubleproteins is retained for further purification of the desired compound.If the product is secreted from desired cells, then the cells areremoved from the culture by low-speed centrifugation, and the supernatefraction is retained for further purification.

The supernatant fraction from either purification method is subjected tochromatography with a suitable resin, in which the desired molecule iseither retained on a chromatography resin while many of the impuritiesin the sample are not, or where the impurities are retained by the resinwhile the sample is not. Such chromatography steps may be repeated asnecessary, using the same or different chromatography resins. Oneskilled in the art would be well-versed in the selection of appropriatechromatography resins and in their most efficacious application for aparticular molecule to be purified. The purified product may beconcentrated by filtration or ultrafiltration, and stored at atemperature at which the stability of the product is maximized.

There is a wide array of purification methods known to the art and thepreceding method of purification is not meant to be limiting. Suchpurification techniques are described, for example, in Bailey, J. E. &Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: New York(1986). Additionally, the identity and purity of the isolated compoundsmay be assessed by techniques standard in the art. These includehigh-performance liquid chromatography (HPLC), spectroscopic methods,staining methods, thin layer chromatography, NIRS, enzymatic assay, ormicrobiologically. Such analysis methods are reviewed in: Patek et al.,1994 Appl. Environ. Microbiol. 60:133-140; Malakhova et al., 1996Biotekhnologiya 11:27-32; and Schmidt et al., 1998 Bioprocess Engineer.19:67-70. Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol.A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566,575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlasof Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.et al. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, vol. 17.

1. A transgenic plant cell transformed with an isolated polynucleotideselected from the group consisting of: a) a polynucleotide having asequence comprising nucleotides 1 to 1347 of SEQ ID NO:8; and b) apolynucleotide encoding a polypeptide having a sequence comprising aminoacids 1 to 218 of SEQ ID NO:15.
 2. The plant cell of claim 1, whereinthe polynucleotide has the sequence comprising nucleotides 1 to 1347 ofSEQ ID NO:8.
 3. The plant cell of claim 1, wherein the polynucleotideencodes the polypeptide having the sequence comprising amino acids 1 to218 of SEQ ID NO:15.
 4. A transgenic plant transformed with an isolatedpolynucleotide selected from the group consisting of: a) apolynucleotide having a sequence comprising nucleotides 1 to 1347 of SEQID NO:8; and b) a polynucleotide encoding a polypeptide having asequence comprising amino acids 1 to 218 of SEQ ID NO:15.
 5. The plantof claim 4, wherein the polynucleotide has the sequence comprisingnucleotides 1 to 1347 of SEQ ID NO:8.
 6. The plant of claim 4, whereinpolynucleotide encodes the polypeptide having the sequence comprisingamino acids 1 to 218 of SEQ ID NO:15.
 7. The plant of claim 4, whereinthe plant is a monocot.
 8. The plant of claim 4, wherein the plant is adicot.
 9. The plant of claim 4, wherein the plant is selected from thegroup consisting of maize, wheat, rye, oat, triticale, rice, barley,soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower,tagetes, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa,coffee, cacao, tea, Salix species, oil palm, coconut, perennial grasses,and a forage crop plant.
 10. A seed which is true breeding for atransgene comprising a polynucleotide selected from the group consistingof: a) a polynucleotide having a sequence comprising nucleotides 1 to1347 of SEQ ID NO:8; and b) a polynucleotide encoding a polypeptidehaving a sequence comprising amino acids 1 to 218 of SEQ ID NO:15. 11.The seed of claim 10, wherein the polynucleotide has the sequencecomprising nucleotides 1 to 1347 of SEQ ID NO:8.
 12. The seed of claim10, wherein the polynucleotide encodes the polypeptide having thesequence comprising amino acids 1 to 218 of SEQ ID NO:15.
 13. Anisolated nucleic acid comprising a polynucleotide selected from thegroup consisting of: a) a polynucleotide having a sequence comprisingnucleotides 1 to 1347 of SEQ ID NO:8; and b) a polynucleotide encoding apolypeptide having a sequence comprising amino acids 1 to 218 of SEQ IDNO:15.
 14. The isolated nucleic acid of claim 13, wherein thepolynucleotide has the sequence comprising nucleotides 1 to 1347 of SEQID NO:8.
 15. The isolated nucleic acid of claim 13, wherein thepolynucleotide encodes the polypeptide having the sequence comprisingamino acids 1 to 218 of SEQ ID NO:15.
 16. A method of producing adrought-tolerant transgenic plant, the method comprising the steps of:a) transforming a plant cell with an expression vector comprising apolynucleotide encoding a polypeptide having a sequence comprising aminoacids 1 to 218 of SEQ ID NO:15; b) growing the transformed plant cell togenerate transgenic plants; and c) screening the transgenic plantsgenerated in step b) to identify a transgenic plant that expresses thepolypeptide and exhibits increased tolerance to drought stress ascompared to a wild type variety of the plant.
 17. The method of claim16, wherein the polynucleotide has a sequence comprising nucleotides 1to 1347 of SEQ ID NO:8.